Identification and treatment of cancer subsets

ABSTRACT

Methods of predicting whether or not a tumor will be responsive to IP6 treatment, methods of treating patients with cancer using IP6, methods of predicting the progression of a disease, and kits that facilitate these methods are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/274,058, filed Oct. 14, 2011 entitled IDENTIFICATION AND TREATMENT OFCANCER SUBSETS, which claims priority to U.S. provisional applicationentitled IDENTIFICATION AND TREATMENT OF CANCER SUBSETS, withapplication No. 61/393,065, filed on 14 Oct. 2010, the contents of theseapplications are hereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA27502 awarded bythe National Institutes of Health. The government has certain rights inthis invention.

FIELD OF THE INVENTION

The present invention is related to skin cancer, and more specifically,methods and kits for skin squamous cell carcinoma prognosis, diagnosis,theranosis and treatment using INPP5A expression as an indication.

BACKGROUND OF THE INVENTION

Over 1,000,000 non-melanoma skin cancers are diagnosed annually in theUS, making these the most common type of cancer and the fifth mostcostly cancer type in the Medicare population. Approximately eightypercent of nonmelanoma skin cancers are basal-cell carcinomas, andtwenty percent are squamous-cell carcinomas (SCC). Unlike almost allbasal-cell carcinomas, cutaneous squamous-cell carcinomas are associatedwith a substantial risk of metastasis. The principal precursor ofcutaneous squamous-cell carcinoma is actinic keratosis (AK). AK has beendescribed as a type of carcinoma in situ or SCCIS, in which carcinomainvolves only the epidermis. Some of AK may evolve into invasivesquamous-cell carcinoma. Options for treating AK include cryosurgery,electrodesiccation and curettage, topical fluorouracil, dermabrasion,and laser resurfacing.

On histological examination actinic keratoses and invasive squamous-cellcarcinomas exhibit a spectrum of neoplastic changes. From a therapeuticstandpoint, it is impractical and unnecessary to treat each individualkeratotic lesion. Only patients with many lesions are followed closely,and thus, many events or tumor progression are undetected until at anadvanced stage. Therefore, accurate prognostic markers and targetedtherapies as well as more effective early chemopreventive strategies arenecessary so that evolving squamous-cell carcinomas can be detected andtreated expeditiously.

BRIEF SUMMARY OF THE INVENTION

Provided herein is a method of identifying a tumor responding to INPP5Ametabolites. The method generally comprises obtaining a sample of thetumor; adding a first reagent to a mixture comprising the sample,wherein the first reagent is capable of detecting a marker comprisingsequence selected from the group consisting of SEQ ID NO. 1 and SEQ IDNO. 2; subjecting the mixture to conditions that allow the detection ofmarker expression; and classifying the tumor into a INPP5Ametabolites-sensitive group based on reduced expression of INPP5A incomparison to a control. In the general method, when the markercomprises SEQ ID NO. 1, the first reagent comprises a first and a secondoligonucleotide capable of binding SEQ ID NO:1; and the method fordetection of the marker is selected from the group consisting of PCR-and hybridization-based methods. In one example, at least one of thefirst and the second oligonucleotides comprises a label comprising afluorescent label selected from the group consisting of FAM, dR110,5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET,BHQ+, Gold540, and LIZ. In this example, the general method may furthercomprise isolating total RNA from the sample; performing reversetranscription of total RNA isolated to obtain cDNA; and subjecting cDNAto conditions that allow nucleic acid amplification by the first andsecond oligonucleotides.

In another example, the first nucleic acid of the general method isaffixed to a substrate; and the method may comprise performingarray-based analysis of SEQ ID NO. 1 to the sample.

In the general method, when the marker comprises SEQ ID NO. 2, the firstreagent comprises a first antibody capable of binding a region of SEQ IDNO. 2. Further, the first antibody comprises a first label selected fromthe group consisting of a fluorescent compound, an enzyme, aradioisotope, and a ligand. In some examples, the general method mayfurther comprise adding a second antibody to the mixture, wherein thesecond antibody is capable of binding to the first antibody. Optionally,the second antibody may comprise a second label selected from the groupconsisting of a fluorescent compound, an enzyme, a radioisotope, and aligand. The general method of detecting the marker may also includedetermining the deletion of INPP5A at chromosome 10q26.3.

The samples in the general method may be in a form selected from thegroup consisting of an FFPE sample, a frozen sample, and a fresh tumorbiopsy. The control in the general method may be selected from the groupconsisting of normal tissue, normal tissue adjacent to the tumor, tissuefrom a less progressed tumor from the same subject. The method mayfurther comprise collecting a sample from a subject selected from agroup consisting of a human, a companion animal, and a livestock animal.The tumor of the sample in the general method may be squamous cellcarcinoma (SCC), squamous-cell carcinoma in situ (SCCIS) or actinickeratosis (AK). Alternatively, the sample in the general method may besuspected to be a skin squamous cell carcinoma (SCC), squamous-cellcarcinoma in situ (SCCIS) or actinic keratosis (AK).

Also provided herein is a method of assessing a risk of progression ofskin carcinoma in a subject, the method comprising: obtaining a skinsample from the patient; adding a reagent capable of binding a markerselected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to amixture comprising the sample; subjecting the mixture to conditions thatallow detection of the marker expression; and labeling the subject ashaving risk of skin carcinoma progression based on the reducedexpression of INPP5A in comparison to a control. Since loss of INPP5Awas shown to be present in a significant percentage of all stages ofSCC, from precursor to metastatic disease, observing loss at AK, SCCIS,or local SCC indicates a risk that the lesion where the loss is observedin may continue to evolve through the stages to eventually becomemetastatic SCC. In this method, if the sample comprises an actinickeratosis, then the progression of the skin carcinoma may compriseprogression to squamous cell carcinoma or metastatic squamous cellcarcinoma. If the sample in the method comprises a squamous cellcarcinoma in situ, then the progression of the skin carcinoma maycomprise progression to squamous cell carcinoma or metastatic squamouscell carcinoma. Alternatively, if the sample comprises a squamous cellcarcinoma, then the progression of the disease comprises progression tometastatic squamous cell carcinoma. The detection of the markerexpression in the general method may be selected from the groupconsisting of PCR-based, hybridization-based, array-based methods andany combinations thereof.

Further provided herein is a method of treating a subject having skinsquamous cell carcinoma at various stages, and the method comprises:obtaining a sample from a subject; determining tumor cell responsivenessto INPP5A metabolites of the sample; administering a pharmaceuticalcomposition comprising one or more INPP5A metabolites or apharmaceutically acceptable salt thereof to the subject; wherein thesubject has tumor cell determined to be responsive to INPP5Ametabolites. In one example, said method may further comprise: adding toa mixture comprising the sample, a reagent capable of binding a markerselected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2;subjecting the mixture to conditions that allow detection of the markerexpression; wherein reduced expression of the marker in comparison to acontrol indicates the cell responsiveness to INPP5A metabolites. Thecontrol of the method is selected from, depending on the stage of thesample tissue, the group consisting of normal skin tissue, normal skintissue adjacent to the tumor of the same subject, skin tissue from aless progressed tumor including actinic keratosis and squamous cellcarcinoma in situ of the same subject. In one example, thepharmaceutical composition of the method comprises IP6 or apharmaceutically acceptable salt thereof, wherein, the pharmaceuticalcomposition of the method causes cessation or reduction of cellproliferation in actinic keratosis, squamous cell carcinoma in situ,squamous cell carcinoma or metastasized squamous cell carcinoma, andrestores cell differentiation resulting in cell death.

In said method, determining the tumor cell responsiveness to INPP5Ametabolites of the sample is a step of detecting the deletion of INPP5Aat chromosome 10q26.3. In some examples, the sample subjected to themethod comprises a skin sample. Further, the sample may comprise a cellselected from the group consisting of actinic keratosis cell; squamouscell carcinoma in situ cell, squamous cell carcinoma cell, andmetastatic squamous cell carcinoma cell.

Further provided herein is a kit for assessing skin tumor responsivenessto INPP5A metabolites. The kit generally comprises: a first reagentcapable of specific detection of a marker selected from the groupconsisting of SEQ ID NO. 1 and SEQ ID NO. 2; and an indicationsignifying a result associated with INPP5A metabolite-sensitivity of thetumor. In one example, the first reagent comprising a first and a secondoligonucleotide capable of binding SEQ ID NO.1, and at least one of thefirst and the second oligonucleotides comprises a label comprising afluorescence moiety or compound selected from the group consisting ofFAM, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED,dROX, PET, BHQ+, Gold540, and LIZ 31. In another example, the firstreagent of the kit comprises a first antibody capable of binding aregion in SEQ ID NO. 2, and the first antibody comprises a first labelselected from the group consisting of a fluorescent compound, an enzyme,a radioisotope, and a ligand. The kit may further comprise a secondantibody capable of binding to the first antibody comprising a secondlabel selected from the group consisting of a fluorescent compound, anenzyme, a radioisotope, and a ligand. The result contained in the kit isbased on the expression level of INPP5A in comparison to a controlselected from the group consisting of normal skin tissue, normal skintissue adjacent to the tumor of the same subject, skin tissue from aless progressed tumor including actinic keratosis and squamous cellcarcinoma in situ of the same subject. In other examples, the kit mayfurther comprise a pharmaceutical composition comprising one or moreINPP5A metabolites or a pharmaceutically acceptable salt thereof. In oneexample, the pharmaceutical composition comprises IP6 or apharmaceutically acceptable salt thereof.

REFERENCE TO COLOR FIGURES

The application file contains at least one figure executed in color.Copies of this patent application publication with color figures will beprovided by the Office upon request and payment of the necessary fee.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the distribution of gene and copy number aberrations in40 tissues examined in this study;

FIG. 2 depicts the identification of selected INPP5A deletions by aCGH;

FIG. 3 depicts loss of INPP5A in a squamous cell carcinoma sample from apatient using blue fluorescence of DAPI bound to DNA (panel labeled as“SCC”) to mark nuclei and chromosomes, green fluorescent probes to markchromosome 10 centromeres, and red fluorescent probes to mark the INPP5Agene near the q-terminus of chromosome 10. A metaphase spread fromnormal cells is shown in panel labeled as “INPP5A Chr10”. The presenceof two copies of the chromosome 10 centromere and two copies of theINPP5A gene in normal skin cells from a patient is shown in panellabeled as “Normal”. The presence of one copy of the chromosome 10centromere and one copy of the INPP5A gene in SCC skin cells from thesame patient is shown in panel labeled as “SCC”;

FIG. 4 depicts the detection of INPP5A protein loss in primary SCCtissues relative to the matched, normal epidermis. Immunohistochemistryfor INPP5A was done on FFPE tissues, and relative intensity of INPP5Astaining (dark brown) was compared between the SCC tissue and theadjacent, histologically normal epidermis. A, bottom, primary skin SCCtissue with low level of INPP5A staining; top, matched, adjacent normalepidermis from the same patient with dark staining in the dermal layer.B, a representative case is shown to further illustrate a relativedifference in INPP5A staining between the primary SCC lesion and theadjacent normal epidermis. Scale bar, 50 μm;

FIG. 5 depicts the detection of INPP5A loss in AK. Immunohistochemistryfor INPP5A was done on FFPE tissues. Two representative lesions areshown in the left and right panel, each containing an area of SCCIS(evident by full epidermal thickness neoplasia; right half of eachimage), arising in association with an AK (partial epidermal thicknessneoplastic change, consistent with AK; left half of each image). Arrowshighlight populations of cells showing low level of INPP5A stainingwithin the AK lesions. Scale bars, 100 μm;

FIG. 6 depicts increased apoptosis in a squamous cell carcinoma cellline that carries a gene expression cassette that expresses INPP5A (Band D) compared to the same cell line carrying a gene expressioncassette that expresses an unrelated protein, green fluorescent protein(A and C). DNA blue fluorescence in (C) and (D) was produced by DAPIprobe, and apoptotic nuclei red fluorescence was produced through TUNELassay;

FIG. 7 depicts the biochemical pathway by which INPP5A directs theproduction of Ins(1,3,4)P3, which is subsequently converted to IP6;

FIG. 8 depicts increased apoptosis in a squamous cell carcinoma cellline treated with IP6 (right panel B) relative to the same line withouttreatment (left panel A), and the DNA blue fluorescence was produced byDAPI probe. Apoptotic nuclei red fluorescence was produced through TUNELassay;

FIG. 9 depicts the fold change of INPP5A mRNA determined by q-RTPCR as afunction of % confluence and differentiation of normal primary humankeratinocytes;

FIG. 10 depicts the change in INPP5A protein expression determined byWestern blot as a function of the % confluence and differentiation ofnormal human keratinocytes;

FIG. 11 depicts the data from FIG. 12 as a graph;

FIG. 12 depicts the effect of IP6 treatment on confluent normal humankeratinocytes. Panel A shows confluent untreated normal humankeratinocytes, while panel B shows confluent normal human keratinocytestreated with IP6. The DNA blue fluorescence was produced by DAPI probe.Apoptotic nuclei red fluorescence was produced through TUNEL assay;

FIG. 13 depicts the effect of IP6 treatment on 70% confluentkeratinocytes. Panel A shows untreated 70% confluent keratinocytes whilepanel B shows 70% confluent keratinocytes treated with IP6. The DNA bluefluorescence was produced by DAPI probe. Apoptotic nuclei redfluorescence was produced through TUNEL assay;

FIG. 14 depicts the plasmid composition of pTUNE vector used to deliverthe INPP5A gene under controlled expression. The coding sequence forINPP5A replaces the GFP, shRNA Target region in the construct used todeliver the INPP5A. The schematic illustrations of the expressionshut-down (A) and expression induction (B) are provided;

FIG. 15 depicts that before and after the introduction of controlledexpression of INPP5A to cell line Cal-27, the production of a smallamount of the protein was sufficient to reduce migration of cells in ascratch assay by 40%. A comparison of a 24 hour assay in the absence(top panels) or presence (bottom panels) of INPP5A in this cell line isshown;

FIG. 16 depicts SCC 15 cells after the INPP5A pTUNE construct wasdelivered by transfection. The nuclei were stained with Vybrant Violet,producing fluorescence in live cells. Normal nuclei show as large, lightgray bodies and apoptosing nuclei appear as small, dark, pycnoticnuclei;

FIG. 17 depicts immunohistochemical staining for the presence of INPP5Awhich shows that it normally appears at a higher and higher level askeratinocytes go through the various stages of differentiation in thetransition from actively growing precursor cells to the dead, cornifiedcells of the outer dermis; and

FIG. 18 depicts IP6 activity against a number of squamous cell cancerlines SCC-4 SCC-15 SCC-9, and both a colorectal adenocarcinoma (HT-29)and a transformed normal human embryonic kidney cell line (HEK-293). Thecells showed a dose dependent reduction in number suggesting thateffects due to INPP5A's loss can to some extent be corrected bysupplying IP6 whose synthesis normally requires the activity of INPP5A.

DETAILED DESCRIPTION OF THE INVENTION

Cutaneous squamous cell carcinoma (SCC) occurs commonly and canmetastasize. Identification of specific molecular aberrations andmechanisms underlying the development and progression of cutaneous SCCcan lead to better prognostic and therapeutic approaches and moreeffective chemoprevention strategies.

A genome-wide survey of gene copy number changes in skin tissuesidentified frequent deletions of INPP5A gene in human SCC tumors. Asprovided herein, a decrease in INPP5A protein levels is observed in mostcutaneous SCCs. This event occurs early in the development of SCC as itcan be detected even at the stage of actinic keratosis, a commonprecursor to SCC. Progressive reduction of INPP5A levels is seen in asubset of SCC patients as the tumor progresses from primary tometastatic stage. Since loss of INPP5A was shown to be present in asignificant percentage of all stages of SCC, from precursor tometastatic disease, observing loss at AK, SCCIS, or local SCC indicatesa risk that the lesion where the loss is observed in may continue toevolve through the stages to eventually become metastatic SCC.

(I) INPP5A as a Gene Marker

Provided herein is a novel gene, INPP5A, the reduced level of which isassociated with development and progression of cutaneous SCC. INPP5A(Type I inositol-1,4,5-trisphosphate 5-phosphatase, UniProtKB/Swiss-ProtAccession Number: Q14642) belongs to a large family of inositolpolyphosphate 5-phosphatases. This 40 kDa membrane-associated type Iinositol phosphatase has preferential substrate affinity for inositol1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5tetrakisphosphate (Ins(1,3,4,5)P4), working mostly as a modulator or themetabolism of inositol phosphates, which are widely used by cells tomodulate and regulate a variety of processes. As used in variouscontexts herein, INPP5A may refer to the nucleic acid form of the gene,or INPP5A may refer to the protein form of the gene.

Further provided herein is the discovery of supplementing inositolphosphate metabolites downstream of the enzyme INPP5A, for example, IP6,for reversal of cancerous cell proliferation to cancer cell death inskin SCC. Therefore, one aspect of the invention provides a method ofidentifying a tumor responsive to INPP5A metabolites. In particular,based on the finding that reduced INPP5A expression, including the mRNAand the protein level, in a subset of skin SCC is associated with thedisease progression as early as AK stage, it has been found that bysupplying at least one of the downstream metabolites of INPP5A to thissubset of cancerous cells, these cells are responsive to theadministration of the metabolite, cancer cell proliferation can beceased, and cell differentiation can be restored, leading to cancer celldeath. In one embodiment, the identified subset of tumor cells withINPP5A deletion is given a supplement of at least one metaboliteselected from the group including Ins(1,3,4)P₃, Ins(1,3,4,6)P₄,Ins(1,3,4,5,6)P₅, and Ins(1,2,3,4,5,6)P₆ (IP6), such that the supplementof INPP5A metabolite leads to cancer cell death. In one preferredembodiment, the identified subset of tumor cell with INPP5A deletion isgiven a supplement of IP6.

Generally, the present invention provides a marker associated with tumorcell's responsiveness to INPP5A metabolites. A marker may be anymolecular structure produced by a cell, expressed inside the cell,accessible on the cell surface, or secreted by the cell. A marker may beany protein, carbohydrate, fatty acid, nucleic acid, catalytic site, orany combination of these such as an enzyme, glycoprotein, cell membrane,virus, a particular cell, or other uni- or multimolecular structure. Amarker may be represented by a sequence of a nucleic acid or any othermolecules derived from the nucleic acid. Examples of such nucleic acidsinclude miRNA, tRNA, siRNA, mRNA, cDNA, genomic DNA sequences, orcomplementary sequences thereof. Alternatively, a marker may berepresented by a protein sequence. The concept of a marker is notlimited to the exact nucleic acid sequence or protein sequence orproducts thereof, rather it encompasses all molecules that may bedetected by a method of assessing the expression of the marker. Withoutbeing limited by the theory, the reduced INPP5A expression, includingthe mRNA and the protein level, in a subset of skin SCC may be caused bydeletion of INPP5A gene, different alleles of INPP5A gene, a mutation inthe INPP5A gene, gene expression regulation abnormally, increased INPP5AmRNA or protein degradation, all of which may result in lower expressionof INPP5A individually or in any combination.

Therefore, examples of molecules encompassed by a marker represented bya particular sequence further include alleles of the gene used as amarker. An allele includes any form of a particular nucleic acid thatmay be recognized as a form of the particular nucleic acid on account ofits location, sequence, or any other characteristic that may identify itas being a form of the particular gene. Alleles include but need not belimited to forms of a gene that include point mutations, silentmutations, deletions, frameshift mutations, single nucleotidepolymorphisms (SNPs), inversions, translocations, heterochromaticinsertions, and differentially methylated sequences relative to areference gene, whether alone or in combination. An allele of a gene mayor may not produce a functional protein; may produce a protein withaltered function, localization, stability, dimerization, orprotein-protein interaction; may have overexpression, underexpression orno expression; may have altered temporal or spatial expressionspecificity. An allele may also be called a mutation or a mutant. Anallele may be compared to another allele that may be termed a wild typeform of an allele. In some cases, the wild type allele is more commonthan the mutant.

A mutation in INPP5A gene that causes decreased activity of INPP5A in atest subject or a biological sample may also be called aloss-of-function mutation. A mutation may be any detectable change ingenetic material such as DNA, or a corresponding change in the RNA orprotein product of that genetic material. A mutant may be any biologicalmaterial in which one or more mutations are detected when compared to acontrol material. Examples of mutations include gene mutations, in whichthe DNA sequence of a gene or any controlling elements surrounding thegene is altered. Controlling elements include promoter, enhancer,suppressor or silencing elements capable of controlling a given gene.Other examples of mutations include alterations in the products of DNAexpression such as RNA or protein that result from correspondingmutations in the DNA. Mutants may also be interchangeably calledvariants. The concept of a mutant includes any change in DNA sequencespecific to the tumor cell (not present in DNA prepared from normal,non-neoplastic tissues).

Loss-of-function mutations display decreased total INPP5A activity inthe test subject or biological sample in comparison with a control,e.g., a healthy subject or a sample without SCC or SCC precursors(standard sample). Therefore, the activity of INPP5A in a subject or asample carrying loss-of-function mutation in INPP5A is 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% relative to that activity ina healthy subject or a standard sample. The decreased activity of INPP5Ain a subject or a sample carrying loss-of-function mutation may resultfrom, for example, decreased basal INPP5A activity, lessened activation,faster degradation, or under-expression, e.g., due to decreased mRNAexpression level, reduced substrate binding, promiscuous orinappropriate substrate binding, impaired recycling resulting inabnormal signaling, increased degradation, or enzyme activation.

A reduced expression level of INPP5A mRNA may result from, for example,a mutation in a non-coding region of a INPP5A gene or a mutation in acoding or non-coding gene involved in INPP5A transcription ortranslation. The expression level of INPP5A can be determined, forexample, by comparing INPP5A mRNA or the level of INPP5A protein in atest subject as compared to a control, for example, by comparing the SCCtumor to normal skin tissue (e.g., a normal adjacent skin tissuesample).

The INPP5A marker provided herein also included conserved variantsencompassing any mutation or other variant in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids withsimilar properties are well known in the art. For example, arginine,histidine and lysine are hydrophilic-basic amino acids and may beinterchangeable. Similarly, isoleucine, a hydrophobic amino acid, may bereplaced with leucine, methionine or valine. Depending on the locationof the mutation in the overall context of the protein, somesubstitutions may have little or no effect on the apparent molecularweight or isoelectric point of the protein or polypeptide. However someconserved variants have been found to alter protein conformation andfunction, including several variants discovered and disclosed herein.

Amino acids other than those indicated as conserved may differ in aprotein or enzyme so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and maybe, for example, from 70% to 99% as determined according to an alignmentscheme such as by the Cluster Method, wherein similarity is based on theMEGALIGN algorithm. The concept of a variant further encompasses apolypeptide or enzyme which has at least 60%, 75%, 85%, 90%, or 95%,amino acid identity as determined by algorithms, such as, BLAST orFASTA, and which has the same or substantially similar properties and/oractivities as the native or parent protein or enzyme to which it iscompared.

One example of such a variant is a loss-of-function variant.Loss-of-function variants of polypeptides encompass any variant in whicha change in one or more amino acid residues in a protein or enzymeimproves the activity of the polypeptide. Examples of activities of apolypeptide that may be impaired by a change resulting in a gain offunction variant include but are not limited to enzymatic activity,binding affinity, phosphorylation or dephosphorylation efficiency,activation or deactivation by a regulatory protein, or any otheractivity or property of a protein that may be quantitatively measured bysome method now known or yet to be disclosed.

Proteins that possess a common evolutionary origin may be homologous orsimilar to one another. Examples of homologous or similar proteinsinclude proteins from superfamilies (e.g., the immunoglobulinsuperfamily) and homologous proteins from different species. Suchproteins and their encoding genes have sequence homology with oneanother. The homology may be expressed in terms of percent similarity orthe presence of specific residues or motifs at conserved positions.

The presence or absence of an allele of the gene marker may be detectedthrough the use of any process known in the art, including for example,using primers and probes designed accordingly for PCR, hybridization,and for some cases, sequencing analyses.

(II) Methods of Detecting Differential Expression of INPP5A in a Sample

A. Method for Identifying Tumor Cells Responsive to INPP5A Metabolites

In one embodiment of the invention, the identification of the INPP5Ametabolite responsive tumor cells generally includes detecting tumorcells with reduced INPP5A mRNA level. In another embodiment of thepresent invention, the identification of the INPP5A metaboliteresponsive tumor cells generally includes detecting tumor cells withreduced INPP5A protein level. In one preferred embodiment, the INPP5Ametabolite is IP6.

Expression of a marker may be assessed by any number of methods used todetect material derived from a nucleic acid template. Differentialexpression of a marker may be assessed or quantified by a detector, aninstrument containing a detector, or by an aided or unaided human eye.Exemplary methods for nucleic acid detection and/or quantificationinclude, but are not limited to, microarray analysis, RNA in situhybridization, RNAse protection assay, Northern blot, reversetranscriptase PCR, quantitative PCR, quantitative reverse transcriptasePCR, quantitative real-time reverse transcriptase PCR, reversetranscriptase treatment followed by direct sequencing, or any othermethod of detecting a specific nucleic acid now known or yet to bedisclosed. Exemplary methods for assessing protein expression include,for example, flow cytometry, immunohistochemistry, ELISA, Western blot,and immunoaffinity chromatograpy, HPLC, mass spectrometry, proteinmicroarray analysis, PAGE analysis, isoelectric focusing, 2-D gelelectrophoresis, or any enzymatic assay. Methods of detecting expressionmay include, for example, methods of purifying nucleic acid, protein, orsome other material depending on a nucleic acid-based or protein-basedapproach. Any method of nucleic acid purification may be used, dependingon the type of marker (i.e., nucleic acid or protein), examples includephenol alcohol extraction, ethanol extraction, guanidium isothionateextraction, gel purification, size exclusion chromatography, cesiumchloride preparations, and silica resin preparation. Any method ofprotein purification may be used, non-limiting examples of which includesize exclusion chromatography, hydrophobic interaction chromatography,ion exchange chromatography, affinity chromatography (including affinitychromatography of tagged proteins), metal binding, immunoaffinitychromatography, and HPLC.

(a) PCR Based Methods

Nucleic acids may be selectively and specifically amplified from atemplate nucleic acid contained in a sample. In some nucleic acidamplification methods, the copies are generated exponentially. Examplesof nucleic acid amplification methods known in the art include:polymerase chain reaction (PCR), ligase chain reaction (LCR),self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification (SDA),amplification with Qβ replicase, whole genome amplification with enzymessuch as φ29, whole genome PCR, in vitro transcription with T7 RNApolymerase or any other RNA polymerase, or any other method by whichcopies of a desired sequence are generated.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA or total RNA to a level detectable by severaldifferent methodologies, for example, hybridization with a labeledprobe, incorporation of biotinylated primers followed by avidin-enzymeconjugate detection, and, incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment.Generally, nucleic acid based probes and primers are complementary to asequence within the target DNA sequence region. In addition to genomicDNA, any oligonucleotide or polynucleotide sequence can be amplifiedwith an appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications.

PCR generally involves the mixing of a nucleic acid sample, two or moreprimers that are designed to recognize the template DNA, a DNApolymerase, which may be a thermostable DNA polymerase such as Taq orPfu, and deoxyribose nucleoside triphosphates (dNTP's). Reversetranscription PCR, quantitative reverse transcription PCR, andquantitative real time reverse transcription PCR are other specificexamples of PCR. In general, the reaction mixture is subjected totemperature cycles comprising a denaturation stage (typically 80-100°C.), an annealing stage with a temperature that is selected based on themelting temperature (Tm) of the primers and the degeneracy of theprimers, and an extension stage (for example 40-75° C.). In real-timePCR analysis, additional reagents, methods, optical detection systems,and devices known in the art are used that allow a measurement of themagnitude of fluorescence in proportion to concentration of amplifiedDNA. In such analyses, incorporation of fluorescent dye into theamplified strands may be detected or measured.

Alternatively, labeled probes that bind to a specific sequence duringthe annealing phase of the PCR may be used with primers. Labeled probesrelease their fluorescent tags during the extension phase so that thefluorescence level may be detected or measured. Generally, probes arecomplementary to a sequence within the target sequence downstream fromeither the upstream or downstream primer. Probes may include one or morelabel. A label may be any substance capable of aiding a machine,detector, sensor, device, or enhanced or unenhanced human eye fromdifferentiating a labeled composition from an unlabeled composition.Examples of labels include but are not limited to: a radioactive isotopeor chelate thereof, dye (fluorescent or nonfluorescent) stain, enzyme,or nonradioactive metal. Specific examples include, but are not limitedto: fluorescein, biotin, digoxigenin, alkaline phosphatese, biotin,streptavidin, ³H, ¹⁴C, ³²P, ³⁵S, or any other compound capable ofemitting radiation, rhodamine, 4-(4′-dimethylamino-phenylazo)benzoicacid (“Dabcyl”); 4-(4′-dimethylamino-phenylazo)sulfonic acid (sulfonylchloride) (“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonicacid (“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives;ethylenediaminetetraacetic acid (“EDTA”) and derivatives thereof or anyother compound that may be differentially detected. The label may alsoinclude one or more fluorescent dyes optimized for use in genotyping.Examples of dyes facilitating the reading of the target amplificationinclude, but are not limited to: CAL-Fluor Red 610, CAL-Fluor Orange560, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED,dROX, PET, BHQ+, Gold540, and LIZ.PCR facilitating the reading of thetarget amplification.

Either primers or primers along with probes allow a quantification ofthe amount of specific template DNA present in the initial sample. Inaddition, RNA may be detected by PCR analysis by first creating a DNAtemplate from RNA through a reverse transcriptase enzyme. The markerexpression may be detected by quantitative PCR analysis facilitatinggenotyping analysis of the samples.

An illustrative example, using dual-labeled oligonucleotide probes inPCR reactions is disclosed in U.S. Pat. No. 5,716,784 to DiCesare. Inone example of the PCR step of the multiplex Real Time-PCR/PCR reactionof the present invention, the dual-labeled fluorescent oligonucleotideprobe binds to the target nucleic acid between the flankingoligonucleotide primers during the annealing step of the PCR reaction.The 5′ end of the oligonucleotide probe contains the energy transferdonor fluorophore (reporter fluor) and the 3′ end contains the energytransfer acceptor fluorophore (quenching fluor). In the intactoligonucleotide probe, the 3′ quenching fluor quenches the fluorescenceof the 5′ reporter fluor. However, when the oligonucleotide probe isbound to the target nucleic acid, the 5′ to 3′ exonuclease activity ofthe DNA polymerase, e.g., Taq DNA polymerase, will effectively digestthe bound labeled oligonucleotide probe during the amplification step.Digestion of the oligonucleotide probe separates the 5′ reporter fluorfrom the blocking effect of the 3′ quenching fluor. The appearance offluorescence by the reporter fluor is detected and monitored during thereaction, and the amount of detected fluorescence is proportional to theamount of fluorescent product released. Examples of apparatus suitablefor detection include, e.g. Applied Biosystems™ 7900HT real-time PCRplatform and Roche's 480 LightCycler, the ABI Prism 7700 sequencedetector using 96-well reaction plates or GENEAMP PC System 9600 or 9700in 9600 emulation mode followed by analysis in the ABA Prism SequenceDetector or TAQMAN LS-50B PCR Detection System. The labeled probefacilitated multiplex Real Time-PCR/PCR can also be performed in otherreal-time PCR systems with multiplexing capabilities.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. Amplification may be a template-specificreplication or a non-template-specific replication (in other words,replication may be specific template-dependent or not). Templatespecificity is here distinguished from fidelity of replication(synthesis of the proper polynucleotide sequence) and nucleotide (ribo-or deoxyribo-) specificity. Template specificity is frequently describedin terms of “target” specificity. Target sequences are “targets” in thesense that they are sought to be sorted out from other nucleic acid.Amplification techniques have been designed primarily for this sortingout.

The term “template” refers to nucleic acid originating from a samplethat is analyzed for the presence of a molecule of interest. Incontrast, “background template” or “control” is used in reference tonucleic acid other than sample template that may or may not be presentin a sample. Background template is most often inadvertent. It may bethe result of carryover, or it may be due to the presence of nucleicacid contaminants sought to be purified out of the sample. For example,nucleic acids from organisms other than those to be detected may bepresent as background in a test sample.

In addition to primers and probes, template specificity is also achievedin some amplification techniques by the choice of enzyme. Amplificationenzymes are enzymes that, under the conditions in which they are used,will process only specific sequences of nucleic acid in a heterogeneousmixture of nucleic acid. Other nucleic acid sequences will not bereplicated by this amplification enzyme. Similarly, in the case of T7RNA polymerase, this amplification enzyme has a stringent specificityfor its own promoters (Chamberlin et al. (1970) Nature (228):227). Inthe case of T4 DNA ligase, the enzyme will not ligate the twooligonucleotides or polynucleotides, where there is a mismatch betweenthe oligonucleotide or polynucleotide substrate and the template at theligation junction (Wu and Wallace (1989) Genomics (4):560). Finally, Taqand Pfu polymerases, by virtue of their ability to function at hightemperature, are found to display high specificity for the sequencesbounded and thus defined by the primers; the high temperature results inthermodynamic conditions that favor primer hybridization with the targetsequences and not hybridization with non-target sequences (H. A. Erlich(ed.) (1989) PCR Technology, Stockton Press).

The term “amplifiable nucleic acid” refers to nucleic acids that may beamplified by any amplification method. It is contemplated that“amplifiable nucleic acid” will usually comprise “sample template.” Theterms “PCR product,” “PCR fragment,” and “amplification product” referto the resultant mixture of compounds after two or more cycles of thePCR steps of denaturation, annealing and extension are complete. Theseterms encompass the case where there has been amplification of one ormore segments of one or more target sequences.

In some forms of PCR assays, quantification of a target in an unknownsample is often required. Such quantification is often in reference tothe quantity of a control sample. The control sample DNA may beco-amplified in the same tube in a multiplex assay or may be amplifiedin a separate tube. Generally, the control sample contains DNA at aknown concentration. The control sample DNA may be a plasmid constructcomprising only one copy of the amplification region to be used asquantification reference. To calculate the quantity of a target in anunknown sample, various mathematical models are established.Calculations are based on the comparison of the distinct cycledetermined by various methods, e.g., crossing points (CP) and cyclethreshold values (Ct) at a constant level of fluorescence; or CPacquisition according to established mathematic algorithm.

The algorithm for Ct values in Real Time-PCR calculates the cycle atwhich each PCR amplification reaches a significant threshold. Thecalculated Ct value is proportional to the number of target copiespresent in the sample, and the Ct value is a precise quantitativemeasurement of the copies of the target found in any sample. In otherwords, Ct values represent the presence of respective target that theprimer sets are designed to recognize. If the target is missing in asample, there should be no amplification in the Real Time-PCR reaction.

Alternatively, the Cp value may be utilized. A Cp value represents thecycle at which the increase of fluorescence is highest and where thelogarithmic phase of a PCR begins. The LightCycler® 480 Softwarecalculates the second derivatives of entire amplification curves anddetermines where this value is at its maximum. By using thesecond-derivative algorithm, data obtained are more reliable andreproducible, even if fluorescence is relatively low.

The various and non-limiting embodiments of the PCR-based methoddetecting marker expression level as described herein may comprise oneor more probes and/or primers. Generally, the probe or primer contains asequence complementary to a sequence specific to a region of the nucleicacid of the marker gene. A sequence having less than 60% 70%, 80%, 90%,95%, 99% or 100% identity to the identified gene sequence may also beused for probe or primer design if it is capable of binding to itscomplementary sequence of the desired target sequence in marker nucleicacid.

(b) Hybridization Based Methods

In addition to PCR, gene expression analysis may also be performed usinga probe that is capable of hybridizing to a nucleic acid sequence ofinterest. The term “hybridization” refers to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e. the strength of the association between the nucleicacids) is impacted by such factors as the degree of complementarybetween the nucleic acids, stringency of the conditions involved, the Tmof the formed hybrid, and the G:C ratio within the nucleic acids. Asingle molecule that contains pairing of complementary nucleic acidswithin its structure is said to be “self-hybridized.”

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, for the sequence “A-G-T,” is complementary to the sequence“T-C-A.” Complementarity may be “partial,” in which only some of thenucleic acids' bases are matched according to the base pairing rules.Or, there may be “complete” or “total” complementarity between thenucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods thatdepend upon binding between nucleic acids.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology, wherein someof the nucleic acids of a first sequence are identical to thecorresponding nucleic acids in a second sequence, or complete homology,wherein the sequences are identical. “Sequence identity” refers to ameasure of relatedness between two or more nucleic acids, and is givenas a percentage with reference to the total comparison length. Theidentity calculation takes into account those nucleotide residues thatare identical and in the same relative positions in their respectivelarger sequences. Calculations of identity may be performed byalgorithms contained within computer programs such as “GAP” (GeneticsComputer Group, Madison, Wis.) and “ALIGN” (DNAStar, Madison, Wis.). Apartially complementary sequence, one that at least partially inhibits(or competes with) a completely complementary sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding, or hybridization, of a sequence that is completelyhomologous to a target under conditions of low stringency. This is notto say that conditions of low stringency are such that non-specificbinding is permitted; low stringency conditions require that the bindingof two sequences to one another be a specific and selective interaction.The absence of non-specific binding may be tested by the use of a secondtarget which lacks even a partial degree of complementarity, forexample, less than about 30% identity. In the absence of non-specificbinding the probe will not hybridize to the second non-complementarytarget.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described infra.

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42° C. when a probe of about 500 nucleotides inlength is employed.

High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

It is well known that numerous equivalent conditions may be employed tocomprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components, forexample, the presence or absence of formamide, dextran sulfate,polyethylene glycol, are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, conditions are known in the art that promote hybridizationunder conditions of high stringency, for example, increasing thetemperature of the hybridization and/or wash steps, the use of formamidein the hybridization solution, etc.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described above.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize,or is the complement of, the single-stranded nucleic acid sequence underconditions of low to high stringency as described above.

The term “Tm” refers to the “melting temperature” of a nucleic acid. Themelting temperature is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half dissociated intosingle strands. The equation for calculating the Tm of nucleic acids iswell known in the art. As indicated by standard references, a simpleestimate of the Tm value may be calculated by the equation:Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 MNaCl (See for example, Anderson and Young, Quantitative FilterHybridization (1985) in Nucleic Acid Hybridization). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of Tm.

As used herein the term “stringency” refers to the conditions oftemperature, ionic strength, and the presence of other compounds such asorganic solvents, under which nucleic acid hybridizations are conducted.With “high stringency” conditions, nucleic acid base pairing will occuronly between nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “low” stringency areoften required with nucleic acids that are derived from organisms thatare genetically diverse, as the frequency of complementary sequences isusually less.

Probes for hybridization may comprise nucleic acids, oligonucleotides(DNA or RNA), proteins, protein complexes, conjugates, natural ligands,small molecules, nanoparticles, or any combination of molecules thatincludes one or more of the above, or any other molecular entity capableof specific binding to any allele, whether such molecular entity existsnow or is yet to be disclosed. In one aspect of the invention, the probecomprises an oligonucleotide, as described above. Other methods used toassess expression include the use of natural or artificial ligandscapable of specifically binding a marker in its protein or peptide form.Such ligands include antibodies, antibody complexes, conjugates, naturalligands, small molecules, nanoparticles, or any other molecular entitycapable of specific binding to a marker. The term “antibody” is usedherein in the broadest sense and refers generally to a molecule thatcontains at least one antigen binding site that immunospecifically bindsto a particular antigen target of interest. The term antibody thusincludes, but is not limited to, native antibodies and variants thereof,fragments of native antibodies and variants thereof, peptibodies andvariants thereof, and antibody mimetics that mimic the structure and/orfunction of an antibody or a specified fragment or portion thereof,including single chain antibodies and fragments thereof. The term thusincludes full length antibodies and/or their variants as well asimmunologically active fragments thereof, thus encompassing antibodyfragments capable of binding to a biological molecule (such as anantigen or receptor) or portions thereof, including but not limited toFab, Fab_, F(ab_)2, facb, pFc_, Fd, Fv or scFv (See, e.g., CURRENTPROTOCOLS IN IMMUNOLOGY, (Colligan et al., eds., John Wiley & Sons,Inc., NY, 1994-2001)).

Ligands may be associated with a label such as a radioactive isotope orchelate thereof, dye (fluorescent or nonfluorescent) stain, enzyme,metal, or any other substance capable of aiding a machine or a human eyefrom differentiating a cell expressing a marker from a cell notexpressing a marker. Additionally, expression may be assessed bymonomeric or multimeric ligands associated with substances capable ofkilling the cell. Such substances include protein or small moleculetoxins, cytokines, pro-apoptotic substances, pore forming substances,radioactive isotopes, or any other substance capable of killing a cell.

In some aspects of the invention, the expression level of a marker genemay be established by binding to probes in a media or on a microarraysuch as a DNA chip. Examples of DNA chips include chips in which anumber of single stranded oligonucleotide probes are affixed to a solidsubstrate such as silicon glass. Oligonucleotides with a sequencecomplementary to an allele are capable of specifically binding to thatallele to the exclusion of alleles that differ from the specific alleleby one or more nucleotides. Labeled sample DNA is hybridized to theoligonucleotides and detection of the label is correlated with bindingof the sample, and consequently, the presence of the allele in thesample.

A nucleic acid probe may be affixed to a substrate. Alternatively, asample may be affixed to the substrate. A probe or sample may becovalently bound to the substrate or it may be bound by some noncovalent interaction including electrostatic, hydrophobic, hydrogenbonding, Van Der Waals, magnetic, or any other interaction by which aprobe such as an oligonucleotide probe may be attached to a substratewhile maintaining its ability to recognize the allele to which it hasspecificity. A substrate may be any solid or semi-solid material ontowhich a probe may be affixed, either singly or in the presence of one ormore additional probes or samples as is exemplified in a microarray.Examples of substrate materials include but are not limited topolyvinyl, polysterene, polypropylene, polyester or any other plastic,glass, silicon dioxide or other silanes, hydrogels, gold, platinum,microbeads, micelles and other lipid formations, nitrocellulose, ornylon membranes. The substrate may take any form, including a sphericalbead or flat surface. For example, the probe may be bound to a substratein the case of an array or an in situ PCR reaction, and the nucleic acidprobe may include a label as described herein.

(c) Sample and Subject

Differential expression encompasses any detectable difference betweenthe expression of a marker in one sample relative to the expression ofthe marker in another sample. Examples include but are not limited todifferential staining of cells in an IHC assay configured to detect amarker, differential detection of bound RNA on a microarray to which asequence capable of binding to the marker is bound, differential resultsin measuring Real Time-PCR measured in the number of PCR cyclesnecessary to reach a particular optical density at a wavelength at whicha double stranded DNA binding dye (e.g., SYBR Green) incorporates,differential results in measuring label from a reporter probe used in areal-time PCR reaction, differential detection of fluorescence on cellsusing a flow cytometer, differential intensities of bands in a Northernblot, differential intensities of bands in an RNAse protection assay,differential cell death measured by apoptotic markers, differential celldeath measured by shrinkage of a tumor, or any method that allows adetection of a difference in signal between one sample or set of samplesand another sample or set of samples.

The expression of the marker in a sample may be compared to a level ofexpression predetermined to predict the presence or absence of aparticular physiological characteristic. The level of expression may bederived from a single control or a set of controls. A control may be anysample with a previously determined level of expression. A control maycomprise material within the sample or material from sources other thanthe sample. Alternatively, the expression of a marker in a sample may becompared to a control that has a level of expression predetermined tosignal or not signal a cellular or physiological characteristic. Thislevel of expression may be derived from a single source of materialincluding the sample itself or from a set of sources. Comparison of theexpression of the marker in the sample to a particular level ofexpression results in a prediction that the sample exhibits or does notexhibit the cellular or physiological characteristic.

The sample in this general tumor identification method is preferably abiological sample from a subject. The term “sample” or “biologicalsample” is used in its broadest sense. Depending upon the embodiment ofthe invention, for example, a sample may comprise a bodily fluidincluding whole blood, serum, plasma, urine, saliva, cerebral spinalfluid, semen, vaginal fluid, pulmonary fluid, tears, perspiration, mucusand the like; an extract from a cell, chromosome, organelle, or membraneisolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution orbound to a substrate; a tissue; a tissue print, or any other materialisolated in whole or in part from a living subject. In one preferredembodiment, the sample is skin tissue. Biological samples may alsoinclude sections of tissues such as biopsy and autopsy samples, andfrozen sections taken for histologic purposes such as blood, plasma,serum, sputum, stool, tears, mucus, hair, skin, and the like. Biologicalsamples also include explants and primary and/or transformed cellcultures derived from patient tissues.

The term “subject” is used in its broadest sense. In a preferredembodiment, the subject is a mammal. Non-limiting examples of mammalsinclude humans, dogs, cats, horses, cows, sheep, goats, and pigs.Preferably, a subject includes any human or non-human mammal, includingfor example: a primate, cow, horse, pig, sheep, goat, dog, cat, orrodent, capable of developing cancer including human patients that aresuspected of having cancer, that have been diagnosed with cancer, orthat have a family history of cancer.

Cancer cells include any cells derived from a tumor, neoplasm, cancer,precancer, cell line, malignancy, or any other source of cells that havethe potential to expand and grow to an unlimited degree. Cancer cellsmay be derived from naturally occurring sources or may be artificiallycreated. Cancer cells may also be capable of invasion into other tissuesand metastasis. Cancer cells further encompass any malignant cells thathave invaded other tissues and/or metastasized. One or more cancer cellsin the context of an organism may also be called a cancer, tumor,neoplasm, growth, malignancy, or any other term used in the art todescribe cells in a cancerous state. In one preferred embodiment, thecancer cell is SCC cell or its precursor Actinic Keratosis cells.

Examples of cancers that could serve as sources of cancer cells includesolid tumors such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, and retinoblastoma. In one preferredembodiment, the cancer is skin cancer.

Additional cancers that may serve as sources of cancer cells includeblood borne cancer, such as acute lymphoblastic leukemia (“ALL,”), acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia (“AML”), acute promyelocytic leukemia(“APL”), acute monoblastic leukemia, acute erythroleukemic leukemia,acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia (“CML”), chronic lymphocytic leukemia (“CLL”), hairycell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenousleukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease,non-Hodgkin's Lymphoma, Waldenstrom's macroglobulinemia, Heavy chaindisease, and Polycythemia vera.

B. Method of Assessing a Risk of Progression of Skin Carcinoma in aSubject.

Another aspect of the invention provides a prognostic method to assessthe risk of a skin SCC precursor to develop into SCC.

Prediction of a cellular or physiological characteristic includes theprediction of any cellular or physiological state that may be predictedby assessing the expression of a marker. Examples include the identityof a cell as a particular cell including a particular normal or cancercell type, the likelihood that one or more diseases is present orabsent, the likelihood that a present disease will progress, remainunchanged, or regress, the likelihood that a disease will respond or notrespond to a particular therapy, or any other outcome. Further examplesinclude the likelihood that a cell will move, senesce, apoptose,differentiate, metastasize, or change from any state to any other stateor maintain its current state.

Expression of a marker in a sample may be more or less than that of alevel predetermined to predict the presence or absence of a cellular orphysiological characteristic. The expression of the marker in the samplemay be more than 1,000,000×, 100,000×, 10,000×, 1000×, 100×, 10×, 5×,2×, 1×, 0.5×, 0.1× 0.01×, 0.001×, 0.0001×, 0.00001×, 0.000001×,0.0000001× or less than that of a level predetermined to predict thepresence or absence of a cellular or physiological characteristic.

The invention contemplates assessing the expression of the marker in anybiological sample from which the expression may be assessed. One skilledin the art would know to select a particular biological sample and howto collect said sample depending upon the marker that is being assessed.Examples of sources of samples include but are not limited to biopsy orother in vivo or ex vivo analysis of prostate, breast, skin, muscle,fascia, brain, endometrium, lung, head and neck, pancreas, smallintestine, blood, liver, testes, ovaries, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow, kidney, placenta, or fetus.In some aspects of the invention, the sample comprises a fluid sample,such as peripheral blood, lymph fluid, ascites, serous fluid, pleuraleffusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimal fluid,stool, or urine. Samples include single cells, whole organs or anyfraction of a whole organ, in any condition including in vitro, ex vivo,in vivo, post-mortem, fresh, fixed, or frozen.

One type of cellular or physiological characteristic is the risk that aparticular disease outcome will occur. Assessing this risk includes theperforming of any type of test, assay, examination, result, readout, orinterpretation that correlates with an increased or decreasedprobability that an individual has had, currently has, or will develop aparticular disease, disorder, symptom, syndrome, or any conditionrelated to health or bodily state. Examples of disease outcomes include,but need not be limited to survival, death, progression of existingdisease, remission of existing disease, initiation of onset of a diseasein an otherwise disease-free subject, or the continued lack of diseasein a subject in which there has been a remission of disease. Assessingthe risk of a particular disease encompasses diagnosis in which the typeof disease afflicting a subject is determined. Assessing the risk of adisease outcome also encompasses the concept of prognosis. A prognosismay be any assessment of the risk of disease outcome in an individual inwhich a particular disease has been diagnosed. Assessing the riskfurther encompasses prediction of therapeutic response in which atreatment regimen is chosen based on the assessment. Assessing the riskalso encompasses a prediction of overall survival after diagnosis.

Determining the level of expression that signifies a physiological orcellular characteristic may be assessed by any of a number of methods.The skilled artisan will understand that numerous methods may be used toselect a level of expression for a particular marker or a plurality ofmarkers that signifies particular physiological or cellularcharacteristics. In diagnosing the presence of a disease, a thresholdvalue may be obtained by performing the assay method on samples obtainedfrom a population of patients having a certain type of disease, forexample, cancer, and from a second population of subjects that do nothave the disease. In assessing disease outcome or the effect oftreatment, a population of patients, all of which have a disease, suchas cancer, may be followed for a period of time. After the period oftime expires, the population may be divided into two or more groups. Forexample, the population may be divided into a first group of patientswhose disease progresses to a particular endpoint and a second group ofpatients whose disease does not progress to the particular endpoint.Examples of endpoints include disease recurrence, death, metastasis orother states to which disease may progress. If expression of the markerin a sample is more similar to the predetermined expression of themarker in one group relative to the other group, the sample may beassigned a risk of having the same outcome as the patient group to whichit is more similar.

In addition, one or more levels of expression of the marker may beselected that signify a particular physiological or cellularcharacteristic. For example, Receiver Operating Characteristic curves,or “ROC” curves, may be calculated by plotting the value of a variableversus its relative frequency in two populations. For any particularmarker, a distribution of marker expression levels for subjects with andwithout a disease may overlap. This indicates that the test does notabsolutely distinguish between the two populations with completeaccuracy. The area of overlap indicates where the test cannotdistinguish the two groups. A threshold is selected. Expression of themarker in the sample above the threshold indicates the sample is similarto one group and expression of the marker below the threshold indicatesthe sample is similar to the other group. The area under the ROC curveis a measure of the probability that the expression correctly indicatedthe similarity of the sample to the proper group. See, e.g., Hanley etal., Radiology 143: 29-36 (1982) hereby incorporated by reference.

Additionally, levels of expression for purpose of prognosis may beestablished by assessing the expression of a marker in a sample from onepatient, assessing the expression of additional samples from the samepatient obtained later in time, and comparing the expression of themarker from the later samples with the initial sample or samples. Thismethod may be used in the case of markers that indicate, for example,progression or worsening of disease or lack of efficacy of a treatmentregimen or remission of a disease or efficacy of a treatment regimen.

Other methods may be used to assess how accurately the expression of amarker signifies a particular physiological or cellular characteristic.Such methods include a positive likelihood ratio, negative likelihoodratio, odds ratio, and/or hazard ratio. In the case of a likelihoodratio, the likelihood that the expression of the marker would be foundin a sample with a particular cellular or physiological characteristicis compared with the likelihood that the expression of the marker wouldbe found in a sample lacking the particular cellular or physiologicalcharacteristic.

An odds ratio measures effect size and describes the amount ofassociation or non-independence between two groups. An odds ratio is theratio of the odds of a marker being expressed in one set of samplesversus the odds of the marker being expressed in the other set ofsamples. An odds ratio of 1 indicates that the event or condition isequally likely to occur in both groups. An odds ratio greater or lessthan 1 indicates that expression of the marker is more likely to occurin one group or the other depending on how the odds ratio calculationwas set up.

A hazard ratio may be calculated by estimate of relative risk. Relativerisk is the chance that a particular event will take place. It is aratio of the probability that an event such as development orprogression of a disease will occur in samples that exceed a thresholdlevel of expression of a marker over the probability that the event willoccur in samples that do not exceed a threshold level of expression of amarker. Alternatively, a hazard ratio may be calculated by the limit ofthe number of events per unit time divided by the number at risk as thetime interval decreases. In the case of a hazard ratio, a value of 1indicates that the relative risk is equal in both the first and secondgroups. A value greater or less than 1 indicates that the risk isgreater in one group or another, depending on the inputs into thecalculation.

Additionally, multiple threshold levels of expression may be determined.This can be the case in so-called “tertile,” “quartile,” or “quintile”analyses. In these methods, multiple groups can be considered togetheras a single population, and are divided into 3 or more bins having equalnumbers of individuals. The boundary between two of these “bins” may beconsidered threshold levels of expression indicating a particular levelof risk of a disease developing or signifying a physiological orcellular state. A risk may be assigned based on which “bin” a testsubject falls into.

C. A Method of Treating a Patient Having at Different Stage of SkinSquamous Cell Carcinoma.

Still another aspect of the present invention provides a method oftreating a patient having skin squamous cell carcinoma or AK cells proneto progress into SCC. The method comprises testing one or more samplesfrom the patient to determine the cell sensitivity to INPP5Ametabolites. If a test is positive, i.e., the cells in the sample havereduced INPP5A expression on either mRNA or protein level, then treatingthe patient with pharmaceutical composition comprising at least oneINPP5A metabolite. In one embodiment, INPP5A metabolite is selected fromthe group consisting of Ins(1,3,4)P₃, Ins(1,3,4,6)P₄, Ins(1,3,4,5,6)P₅,and Ins(1,2,3,4,5,6)P₆ (IP6). In one preferred example, the INPP5Ametabolite is IP6 In one preferred embodiment, the pharmaceuticalcomposition comprises IP6.

In general, the pharmaceutical composition will comprise an effectivedosage amount of the disclosed one or more INPP5A metabolites, i.e., anamount of INPP5A metabolites sufficient to provide treatment to thesubject being administered the pharmaceutical composition. Determinationof an effective amount of the composition is within the capability ofthose skilled in the art. The effective amount of a pharmaceuticalcomposition used to affect a particular purpose, as well as itstoxicity, excretion, and overall tolerance may be determined in cellcultures or experimental animals by pharmaceutical and toxicologicalprocedures either known now by those skilled in the art or by anysimilar method yet to be disclosed. One example is the determination ofthe IC₅₀ (half maximal inhibitory concentration) of the pharmaceuticalcomposition in vitro in cell lines or target molecules. Another exampleis the determination of the LD₅₀ (lethal dose causing death in 50% ofthe tested animals) of the pharmaceutical composition in experimentalanimals. The exact techniques used in determining an effective amountwill depend on factors such as the type, physical and/or chemicalproperties of the pharmaceutical composition, the property being tested,and whether the test is to be performed in vitro or in vivo. Thedetermination of an effective amount of a pharmaceutical compositionwill be well known to one of skill in the art who will use data obtainedfrom any tests in making that determination. Determination of aneffective amount of compound for addition to a cancer cell also includesthe determination of an effective therapeutic amount, including theformulation of an effective dose range for use in vivo, including inhumans.

In some embodiments, the pharmaceutical composition may comprisesubstantially pure INPP5A metabolite. In one preferred embodiment, theINPP5A metabolite is IP6 or a pharmaceutically acceptable salt thereof.The amount of INPP5A IP6 or a pharmaceutically acceptable salt thereofin such pharmaceutical compositions, therefore, may range from about97%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, orabout 3% by weight of the total amount of IP6 and its salt.

A variety of excipients commonly used in pharmaceutical formulations maybe selected on the basis of several criteria, such as, the desireddosage form and the release profile properties of the dosage form.Non-limiting examples of suitable excipients include an agent selectedfrom the group consisting of a binder, a filler, a non-effervescentdisintegrant, an effervescent disintegrant, a preservative, a diluent, aflavoring agent, a sweetener, a lubricant, an oral dispersing agent, acoloring agent, a taste masking agent, a pH modifier, a stabilizer, acompaction agent, and combinations of any of these agents.

In one embodiment, the excipient may be a binder. Suitable bindersinclude starches, pregelatinized starches, gelatin, polyvinylpyrolidone,cellulose, methylcellulose, sodium carboxymethylcellulose,ethylcellulose, polyacrylamides, polyvinyloxoazolidone,polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol,polyols, saccharides, oligosaccharides, polypeptides, peptides, andcombinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillersinclude carbohydrates, inorganic compounds, and polyvinylpirrolydone. Byway of non-limiting example, the filler may be calcium sulfate, both di-and tri-basic, starch, calcium carbonate, magnesium carbonate,microcrystalline cellulose, dibasic calcium phosphate, magnesiumcarbonate, magnesium oxide, calcium silicate, talc, modified starches,lactose, sucrose, mannitol, and sorbitol.

The excipient may be a non-effervescent disintegrant. Suitable examplesof non-effervescent disintegrants include starches (such as corn starch,potato starch, and the like), pregelatinized and modified starchesthereof, sweeteners, clays (such as bentonite), microcrystallinecellulose, alginates, sodium starch glycolate, and gums (such as agar,guar, locust bean, karaya, pecitin, and tragacanth).

In another embodiment, the excipient may be an effervescentdisintegrant. By way of non-limiting example, suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

The excipient may comprise a preservative. Suitable examples ofpreservatives include antioxidants (such as alpha-tocopherol orascorbate) and antimicrobials (such as parabens, chlorobutanol orphenol). In other embodiments, an antioxidant such as butylatedhydroxytoluene (BHT) or butylated hydroxyanisole (BHA) may be utilized.

In another embodiment, the excipient may include a diluent. Diluentssuitable for use include pharmaceutically acceptable saccharides such assucrose, dextrose, lactose, microcrystalline cellulose, fructose,xylitol, and sorbitol; polyhydric alcohols; starches; pre-manufactureddirect compression diluents; and mixtures of any of the foregoing.

The excipient may include flavoring agents. Flavoring agents may bechosen from synthetic flavor oils and flavoring aromatics and/or naturaloils, extracts from plants, leaves, flowers, fruits, and combinationsthereof. By way of example, these may include cinnamon oils, oil ofwintergreen, peppermint oils, clover oil, hay oil, anise oil,eucalyptus, vanilla, citrus oils (such as lemon oil, orange oil, grapeand grapefruit oil), and fruit essences (such as apple, peach, pear,strawberry, raspberry, cherry, plum, pineapple, and apricot).

In another embodiment, the excipient may include a sweetener. By way ofnon-limiting example, the sweetener may be selected from glucose (cornsyrup), dextrose, invert sugar, fructose, and mixtures thereof (when notused as a carrier); saccharin and its various salts such as the sodiumsalt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; stevia-derived sweeteners; chloro derivatives of sucrosesuch as sucralose; sugar alcohols such as sorbitol, mannitol, sylitol,and the like. Also contemplated are hydrogenated starch hydrolysates andthe synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In another embodiment, the excipient may be a lubricant. Suitablenon-limiting examples of lubricants include magnesium stearate, calciumstearate, zinc stearate, hydrogenated vegetable oils, sterotex,polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate,sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.

The excipient may be a dispersion enhancer. Suitable dispersants mayinclude starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose.

Depending upon the embodiment, it may be desirable to provide a coloringagent. Suitable color additives include food, drug and cosmetic colors(FD&C), drug and cosmetic colors (D&C), or external drug and cosmeticcolors (Ext. D&C). These colors or dyes, along with their correspondinglakes, and certain natural and derived colorants may be suitable for usein the present invention depending on the embodiment.

The excipient may include a taste-masking agent. Taste-masking materialsinclude cellulose hydroxypropyl ethers (HPC); low-substitutedhydroxypropyl ethers (L-HPC); cellulose hydroxypropyl methyl ethers(HPMC); methylcellulose polymers and mixtures thereof; polyvinyl alcohol(PVA); hydroxyethylcelluloses; carboxymethylcelluloses and saltsthereof; polyvinyl alcohol and polyethylene glycol co-polymers;monoglycerides or triglycerides; polyethylene glycols; acrylic polymers;mixtures of acrylic polymers with cellulose ethers; cellulose acetatephthalate; and combinations thereof.

In various embodiments, the excipient may include a pH modifier. Incertain embodiments, the pH modifier may include sodium carbonate orsodium bicarbonate.

The weight fraction of the excipient or combination of excipients in thepharmaceutical composition may be about 98% or less, about 95% or less,about 90% or less, about 85% or less, about 80% or less, about 75% orless, about 70% or less, about 65% or less, about 60% or less, about 55%or less, about 50% or less, about 45% or less, about 40% or less, about35% or less, about 30% or less, about 25% or less, about 20% or less,about 15% or less, about 10% or less, about 5% or less, about 2%, orabout 1% or less of the total weight of the pharmaceutical composition.

The pharmaceutical compositions detailed herein may be manufactured inone or several dosage forms. Suitable dosage forms include transdermalsystems or patches. The transdermal system may be a matrix system, areservoir system, or a system without rate-controlling membranes. Othersuitable dosage forms also include tablets, including suspensiontablets, chewable tablets, effervescent tablets or caplets; pills;powders such as a sterile packaged powder, a dispensable powder, and aneffervescent powder; capsules including both soft or hard gelatincapsules such as HPMC capsules; lozenges; a sachet; a sprinkle; areconstitutable powder or shake; a troche; pellets such as sublingual orbuccal pellets; granules; liquids for oral or parenteral administration;suspensions; emulsions; semisolids; or gels.

The dosage forms may be manufactured using conventional pharmacologicaltechniques. Conventional pharmacological techniques include, e.g., oneor a combination of methods: (1) dry mixing, (2) direct compression, (3)milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6)fusion. See, e.g., Lachman et al., The Theory and Practice of IndustrialPharmacy (1986). Other methods include, e.g., prilling, spray drying,pan coating, melt granulation, granulation, wurster coating, tangentialcoating, top spraying, extruding, coacervation and the like.

The amount of active ingredient that is administered to a subject canand will vary depending upon a variety of factors such as the age andoverall health of the subject, and the particular mode ofadministration. Those skilled in the art will appreciate that dosagesmay also be determined with guidance from Goodman & Goldman's ThePharmacological Basis of Therapeutics, Tenth Edition (2001), AppendixII, pp. 475-493, and the Physicians' Desk Reference. In one embodimentof the present invention, the effective amount of the disclosed compoundto results in the slowing of expansion of the cancer cells wouldpreferably result in a concentration at or near the target tissue thatis effective in slowing cellular expansion in cancer cells, but haveminimal effects on non-cancer cells, including non-cancer cells exposedto radiation or recognized chemotherapeutic chemical agents.Concentrations that produce these effects can be determined using, forexample, apoptosis markers such as the apoptotic index and/or caspaseactivities either in vitro or in vivo. In one preferred embodiment, theeffective dosage results in the resumed differentiation of SCC cell orits precursor AK cell, reduced cell proliferation of cancerous cell orits precursor and increased cancerous cell death.

Methods of administration include, but are not limited to, oraladministration and parenteral administration. Parenteral administrationincludes, but is not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,sublingual, intramsal, intracerebral, iratraventricular, intrathecal,intravaginal, transdermal, rectal, by inhalation, or topically to theears, nose, eyes, or skin. Other methods of administration include butare not limited to infusion techniques including infusion or bolusinjection, by absorption through epithelial or mucocutaneous liningssuch as oral mucosa, rectal and intestinal mucosa. Compositions forparenteral administration may be enclosed in ampoule, a disposablesyringe or a multiple-dose vial made of glass, plastic or othermaterial.

Administration may be systemic or local. Local administration isadministration of the disclosed compound to the area in need oftreatment. Examples include local infusion during surgery; topicalapplication, by local injection; by a catheter; by a suppository; or byan implant. Administration may be by direct injection at the site (orformer site) of a cancer, tumor, or precancerous tissue or into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection. Intraventricular injection can be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration may be achieved byany of a number of methods known in the art. Examples include use of aninhaler or nebulizer, formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Thedisclosed compound may be delivered in the context of a vesicle such asa liposome or any other natural or synthetic vesicle.

A pharmaceutical composition formulated so as to be administered byinjection may be prepared by dissolving the disclosed compound withwater so as to form a solution. In addition, a surfactant may be addedto facilitate the formation of a homogeneous solution or suspension.Surfactants include any complex capable of non-covalent interaction withthe disclosed compound so as to facilitate dissolution or homogeneoussuspension of the compound.

Pharmaceutical compositions including the disclosed compound may beprepared in a form that facilitates topical or transdermaladministration. Such preparations may be in the form of a liquidsolution, cream, paste, lotion, shake lotion, powder, emulsion,ointment, gel base, transdermal patch or iontophoresis device. Examplesof bases used in such compositions include petrolatum, lanolin,polyethylene glycols, beeswax, mineral oil, diluents such as water andalcohol, and emulsifiers and stabilizers, thickening agents, or anyother suitable base now known or yet to be disclosed.

Addition of a pharmaceutical composition to cancer cells includes allactions by which an effect of the pharmaceutical composition on thecancer cell is realized. The type of addition chosen will depend uponwhether the cancer cells are in vivo, ex vivo, or in vitro, the physicalor chemical properties of the pharmaceutical composition, and the effectthe composition is to have on the cancer cell. Nonlimiting examples ofaddition include addition of a solution including the pharmaceuticalcomposition to tissue culture media in which in vitro cancer cells aregrowing; any method by which a pharmaceutical composition may beadministered to an animal including intravenous, per os, parenteral, orany other of the methods of administration; or the activation orinhibition of cells that in turn have effects on the cancer cells suchas immune cells (e.g. macophages and CD8+ T cells) or endothelial cellsthat may differentiate into blood vessel structures in the process ofangiogenesis or vasculogenesis.

Treatment is contemplated in living entities including but not limitedto mammals (particularly humans) as well as other mammals of economic orsocial importance, including those of an endangered status. Furtherexamples include livestock or other animals generally bred for humanconsumption and domesticated companion animals. Treatment of a conditionis the practice of any method, process, or procedure with the intent ofhalting, inhibiting, slowing or reversing the progression of a disease,disorder or condition, substantially ameliorating clinical symptoms of adisease disorder or condition, or substantially preventing theappearance of clinical symptoms of a disease, disorder or condition, upto and including returning the diseased entity to its condition prior tothe development of the disease.

The method of treating a subject having a form of cancer includes theprevention of progression of the cancer to a neoplastic, malignant ormetastatic state. Such preventative use is indicated in conditions knownor suspected of preceding progression to cancer, in particular, wherenon- or precancerous cell growth consisting of hyperplasia, metaplasia,or most particularly, dysplasia has occurred (for review of suchabnormal growth conditions, see Robbins and Angell, 1976, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-90,incorporated by reference). Hyperplasia is a form of controlled cellproliferation involving an increase in cell number in a tissue or organ,without significant alteration in structure or activity. For example,endometrial hyperplasia often precedes endometrial cancer andprecancerous colon polyps often transform into cancerous lesions.Metaplasia is a form of controlled cell growth in which one type ofadult or fully differentiated cell substitutes for another type of adultcell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplasticepithelium. Dysplasia is frequently a forerunner of cancer, and is foundmainly in the epithelia; it is the most disorderly form ofnon-neoplastic cell growth, involving a loss in individual celluniformity and in the architectural orientation of cells. Dysplasticcells often have abnormally large, deeply stained nuclei, and exhibitpleomorphism.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype or of a malignantphenotype, displayed in vivo or displayed in vitro by a cell samplederived from a subject can indicate the desirability ofprophylactic/therapeutic administration of the pharmaceuticalcomposition that includes the compound. Such characteristics of atransformed phenotype include morphology changes, looser substratumattachment, loss of contact inhibition, loss of anchorage dependence,protease release, increased sugar transport, decreased serumrequirement, expression of fetal antigens, disappearance of the 250,000dalton cell surface protein, etc. In a preferred embodiment, thecharacteristics of a transformed phenotype due to the treatment usingpharmaceutical composition comprising INPP5A metabolite(s) is thedifferentiation of SCC cell or its precursor AK cell that leads to thecancerous cell death.

Pharmaceutical compositions that include the disclosed compound may beadministered prior to, concurrently with, or after administration of asecond pharmaceutical composition that may or may not include thecompound. If the compositions are administered concurrently, they areadministered within one minute of each other. If not administeredconcurrently, the second pharmaceutical composition may be administereda period of one or more minutes, hours, days, weeks, or months before orafter the pharmaceutical composition that includes the compoundAlternatively, a combination of pharmaceutical compositions may becyclically administered. Cycling therapy involves the administration ofone or more pharmaceutical compositions for a period of time, followedby the administration of one or more different pharmaceuticalcompositions for a period of time and repeating this sequentialadministration, in order to reduce the development of resistance to oneor more of the compositions, to avoid or reduce the side effects of oneor more of the compositions, and/or to improve the efficacy of thetreatment.

The invention further encompasses methods of treating cancer thatcomprise combination therapies that comprise the administration of apharmaceutical composition including the disclosed compound and anothertreatment modality. Such treatment modalities include but are notlimited to, radiotherapy, chemotherapy, surgery, immunotherapy, cancervaccines, radioimmunotherapy, treatment with pharmaceutical compositionsother than those which include the disclosed compound, or any othermethod that effectively treats cancer in combination with the disclosedcompound now known or yet to be disclosed. Combination therapies may actsynergistically. That is, the combination of the two therapies is moreeffective than either therapy administered alone. This results in asituation in which lower dosages of both treatment modality may be usedeffectively. This in turn reduces the toxicity and side effects, if any,associated with the administration either modality without a reductionin efficacy.

In one preferred embodiment, the cancers or precancers that may betreated by pharmaceutical compositions including the disclosed compoundeither alone or in combination with another treatment modality is skinSCC.

(III) Kits.

The present invention further provides kits to be used in assessing theexpression of a particular RNA in a sample from a subject to assess therisk of developing disease. Kits include any combination of componentsthat facilitate the performance of an assay. A kit that facilitatesassessing the expression of a RNA may include suitable nucleicacid-based and immunological reagents as well as suitable buffers,control reagents, and printed protocols.

Kits that facilitate nucleic acid based methods may further include oneor more of the following: specific nucleic acids such asoligonucleotides, labeling reagents, enzymes including PCR amplificationreagents such as Taq or Pfu; reverse transcriptase, or one or more otherpolymerases, and/or reagents that facilitate hybridization. Specificnucleic acids may include nucleic acids, polynucleotides,oligonucleotides (DNA, or RNA), or any combination of molecules thatincludes one or more of the above, or any other molecular entity capableof specific binding to a nucleic acid marker. In one aspect of theinvention, the specific nucleic acid comprises one or moreoligonucleotides capable of hybridizing to the marker.

A specific nucleic acid may include a label. A label may be anysubstance capable of aiding a machine, detector, sensor, device, orenhanced or unenhanced human eye from differentiating a sample that thatdisplays positive expression from a sample that displays reducedexpression. Examples of labels include but are not limited to: aradioactive isotope or chelate thereof, a dye (fluorescent ornonfluorescent) stain, enzyme, or nonradioactive metal. Specificexamples include but are not limited to: fluorescein, biotin,digoxigenin, alkaline phosphatase, biotin, streptavidin, ³H, ¹⁴C, ³²P,³⁵S, or any other compound capable of emitting radiation, rhodamine,4-(4′-dimethylaminophenylazo)benzoic acid (“Dabcyl”);4-(4′-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)(“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid(“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives;ethylenediaminetetraaceticacid (“EDTA”) and derivatives thereof or anyother compound that signals the presence of the labeled nucleic acid. Inone embodiment of the invention, the label includes one or more dyesoptimized for use in genotyping. Examples of such dyes include but arenot limited to: dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA,TAMRA, NED, dROX, PET, and LIZ.

An oligonucleotide is a reagent capable of binding a nucleic acidsequence. An oligonucleotide may be any polynucleotide of at least 2nucleotides. Oligonucleotides may be less than 10, less than 15, lessthan 20, less than 30, less than 40, less than 50, less than 75, lessthan 100, less than 200, less than 500, or more than 500 nucleotides inlength. While oligonucleotides are often linear, they may, depending ontheir sequence and conditions, assume a two- or three-dimensionalstructure. Oligonucleotides may be chemically synthesized by any of anumber of methods including sequential synthesis, solid phase synthesis,or any other synthesis method now known or yet to be disclosed.Alternatively, oligonucleotides may be produced by recombinant DNA basedmethods. One skilled in the art would understand the length ofoligonucleotide necessary to perform a particular task. Oligonucleotidesmay be directly labeled, used as primers in PCR or sequencing reactions,or bound directly to a solid substrate as in oligonucleotide arrays.

An oligonucleotide used to detect to an allele may be affixed to a solidsubstrate. Alternatively, the sample may be affixed to a solid substrateand the nucleic acid reagent placed into a mixture. For example, thenucleic acid reagent may be bound to a substrate in the case of an arrayor the sample may be bound to a substrate as the case of a SouthernBlot, Northern blot or other method that affixes the sample to asubstrate. A nucleic acid reagent or sample may be covalently bound tothe substrate or it may be bound by some non covalent interactionincluding electrostatic, hydrophobic, hydrogen bonding, Van Der Waals,magnetic, or any other interaction by which an oligonucleotide may beattached to a substrate while maintaining its ability to recognize theallele to which it has specificity. A substrate may be any solid or semisolid material onto which a probe may be affixed, attached or printed,either singly or in the formation of a microarray. Examples of substratematerials include but are not limited to polyvinyl, polysterene,polypropylene, polyester or any other plastic, glass, silicon dioxide orother silanes, hydrogels, gold, platinum, microbeads, micelles and otherlipid formations, nitrocellulose, or nylon membranes. The substrate maytake any shape, including a spherical bead or flat surface. In someaspects of the invention, the probe may be affixed to a solid substrate.In other aspects of the invention, the sample may be affixed to a solidsubstrate.

Kits may also contain reagents that detect proteins, often through theuse of an antibody. These kits will contain one or more specificantibodies, buffers, and other reagents configured to detect binding ofthe antibody to the specific epitope. One or more of the antibodies maybe labeled with a fluorescent, enzymatic, magnetic, metallic, chemical,or other label that signifies and/or locates the presence ofspecifically bound antibody. The kit may also contain one or moresecondary antibodies that specifically recognize epitopes on otherantibodies. These secondary antibodies may also be labeled. The conceptof a secondary antibody also encompasses non-antibody ligands thatspecifically bind an epitope or label of another antibody. For example,streptavidin or avidin may bind to biotin conjugated to anotherantibody. Such a kit may also contain enzymatic substrates that changecolor or some other property in the presence of an enzyme that isconjugated to one or more antibodies included in the kit.

A kit may also contain an indication of a result of the use of the kitthat signifies a particular physiological or cellular characteristic. Anindication includes any guide to a result that would signal the presenceor absence of any physiological or cellular state that the kit isconfigured to predict. For example, the indication may be expressednumerically, expressed as a color or density of a color, expressed as anintensity of a band, derived from a standard curve, or expressed incomparison to a control. The indication may be communicated through theuse of a writing. A writing may be any communication of the result in atangible medium of expression. The writing may be contained physicallyin or on the kit (on a piece of paper for example), posted on theInternet, mailed to the user separately from the kit, or embedded in asoftware package. The writing may be in any medium that communicates howthe result may be used to predict the cellular or physiologicalcharacteristic that the kit is intended to predict, such as a printeddocument, a photograph, sound, color, or any combination thereof.

The invention further encompasses pharmaceutical compositions thatinclude the disclosed compound and/or pharmaceutically acceptable saltsof the compound.

EXAMPLES

Various embodiments of the present teachings can be illustrated by thefollowing non-limiting examples. The following examples areillustrative, and are not intended to limit the scope of the claims.

Methods and Material:

Tissues:

Tissue samples analyzed were formalin-fixed, paraffin-embedded (FFPE),archived specimens obtained under the Institutional ReviewBoard-approved protocols at the Arizona Cancer Center, University ofArizona (Tucson, Ariz.); Southern Arizona Veterans Affairs Health CareSystem (Tucson, Ariz.); Loyola University Medical Center (Chicago,Ill.); and Mayo Clinic. The study was conducted according to theDeclaration of Helsinki Principles.

Array CGH:

To obtain genomic DNA for aCGH (array-based comparative genomichybridization), microscopic examination by pathologist was used toselect the areas for harvest and DNA extraction. In all samples, onlyregions that showed >50% lesional content were harvested. aCGH profilingwas done using a method in which DNA was extracted from FFPE tissueblocks using the DNeasy tissue kit (Qiagen, Germantown, Md.). Normalpooled lymphocyte DNA (Promega, Madison, Wis.) was used as a reference.A total of 5 μg of sample genomic DNA and 1 μg of reference genomic DNAwere fragmented using the thermolabile recombinant shrimp DNase(TS-DNase; Affymetrix, Santa Clara, Calif.) to achieve an average DNAfragment length of 200 to 600 bp. Fragmented sample and reference DNAwere labeled with Cy5 and Cy3 fluorescent dUTP, respectively, using theBioprime Array CGH Genomic Labeling System (Invitrogen, Carlsbad,Calif.). Hybridizations were done on Agilent 44K feature microarrays foraCGH (Agilent Technologies, Santa Clara, Calif.) per the manufacturer'sspecifications and scanned on an Agilent DNA Microarray scanner,followed by image analysis with Feature Extraction software and datavisualization with DNA Analytics software using the aberration callingalgorithm ADM-1.

Fluorescence In Situ Hybridization:

Fluorescence in situ hybridization (FISH) was carried out using acentromeric probe to chromosome 10 (Abbott Molecular, Des Plaines, Ill.)and INPP5A-directed probes (bacterial artificial chromosomes RP11-500B2and RP11-288G11; BACPAC Resource Center) to either metaphase spreads orsections prepared from FFPE blocks. The DNA from two large plasmidscarrying a large segment of human chromosomal DNA in the INPP5A generegion (RP11-500B2 and RP11-288G11) was isolated and labeled with Cy5tagged nucleotides, then hybridized to the FFPE slices from normal andSCC samples. FFPE slices were prepared for hybridization using theParaffin Pretreatment Kit II (Abbott Molecular, Des Plaines, Ill.).Slides were examined and photographed on a Zeiss Axiophot equipped withinterference filters (Chroma, Bellows Falls, Vt.) and a CoolSnap HQ2digital camera (Photometrics, Tucson, Ariz.). The FISH evaluation wassemiquantitative. Whenever the tissue was of sufficient size, 100+nuclei were examined. However, in cases where a lesion of interest wassmall (e.g., AK lesions), all available lesional nuclei (i.e., <100)were examined.

Immunohistochemistry:

FFPE tissue blocks were sectioned on glass slides at 5-μm thickness andbaked for 60 minutes at 60° C. Slides were subsequently subjected toheat-induced epitope retrieval using a proprietary citrate-basedretrieval solution for 20 minutes. The tissue sections were incubatedfor 30 minutes with anti-INPP5A mouse monoclonal antibody (clone 3D8;Novus Biologicals, Littleton, Colo.). The sections were visualized withthe Bond Polymer Refine Detection kit (Leica Microsystems, Inc.,Wetzlar, Germany) using diaminobenzidine chromogen as substrate.

IP6 Treatment of Squamous Cell Carcinoma:

The human head and neck squamous cell carcinoma lines evaluated were:SCC-4, SCC-9 and SCC-15. The colorectal cancer cell line HT-29 and thekidney embryonic line HEK293, purchased from the American Type CultureCollection (ATCC, Manassas, Va.), were used for comparison. Squamouscell lines were maintained in Dulbecco's Modified Eagle Medium: NutrientMixture F12 (DMEM/F12), with 10% Fetal bovine serum (FBS), 2 mMGlutaMAX, 25 mM Hepes buffer, 100 U/ml penicillin and 100 μg/mlstreptomycin. HEK293and HT-29 were cultured in DMEM and RPMI-1640 mediumrespectively, with the supplements mentioned above. All media and cellculture supplements were purchased from Invitrogen Corporation(Carlsbad, Calif.). Sub-confluent, rapidly growing cells were plated ata density of 600 cells/20 μL/well in a clear bottom 384-well plate withopaque walls, suitable for luminescent measurements. The next day, IP6(Inositol hexakisphosphate, phytic acid, dipotassium salt,Sigma-Aldrich, St Louis, Mo.) solutions in culture medium, correspondingto the following concentrations: 0.3125, 0.625, 1.25, 2.5 and 5 mM, wereprepared, and were stored at 4° C. for 4-6 h. Shortly before treatment,IP6 solutions were centrifuged to remove any precipitates that IP6 formswith calcium chloride. Four wells were treated for each condition. 48 hafter IP6 treatments, cells were assayed for viability using theCellTiter-Glo® Luminescent Cell Viability Assay kit, fromPromegaBioSciences, Inc. (San Luis Obispo, Calif.), following themanufacturer's instruction. Briefly, the assay buffer and substrate wereequilibrated at room temperature, and mixed thoroughly. Without removingmedium from wells, the assay reagent was added into each well (1:1,volume:volume of medium per well) and the content was mixed for 2minutes on an orbital shaker to induce cell lysis. The plate wasincubated at room temperature for 10 minutes, and the luminescence wasread on a Perkin Elmer Victor³microplatereader. Data from each set offour replicates was averaged and the standard deviation was calculated.

Statistics:

The two-tailed Fisher's exact test was used to compare the stainingpatterns between the cohorts of primary SCC tumors that havesubsequently metastasized with those that have not. P values of <0.05were considered statistically significant.

Example 1 The INPP5A Gene is Frequently Deleted in Cutaneous SCC Tumors

To identify novel genes and molecular mechanisms associated withcutaneous SCC (squamous cell carcinoma) development and progression, aseries of archived skin tissues spanning a range from normal skin toinvasive SCC were analyzed. An optimized high-resolution oligomer aCGHmethod was used on these archived FFPE tissues. This approach enableddetecting gene copy number changes with sensitivity and accuracycomparable with that obtained by analysis of DNA derived from frozentissues.

aCGH was done using the DNA from a spectrum of 40 FFPE skin tissues,including normal skin (n=12), precancerous lesions of AK (n=5), in situSCC lesions (SCCIS; n=2), and invasive SCCs (SCC; n=21). A total of 458copy number aberrations were identified in the examined samples, 267(58%) of which were amplifications and 191 (42%) were deletions. It wasobserved that there was an increase in the overall frequency of genecopy number aberrations per sample in proportion to the increasingmalignant characteristics of the examined tissue, with invasive SCCsharboring, on average, the highest number of aberrations per genome(FIG. 1).

Examination of the genomic regions characterized by recurrent copynumber changes across samples, as detected by aCGH, identified aprevalent copy number aberration which was deletion of the q-ter regionof chromosome 10, an area harboring the INPP5A (inositol polyphosphate5-phosphatases) gene (FIG. 2). aCGH detected loss of the INPP5A gene in1 of 2 examined SCCIS lesions, and in 5 of 21 (24%) examined invasiveSCC tumors, but in none of the examined AK lesions or normal skin (Table1).

TABLE 1 Frequency of INPP5A gene deletions as detected by CGH Numberwith INPP5A Tissue Type deletion by aCGH Normal  0/12 AK 0/5 SCCIS 1/2SCC  5/21

To verify the accuracy of aCGH calls, FISH (fluorescent in situhybridization) for INPP5A was performed in two of five samples thatshowed INPP5A deletions by aCGH. Both cases showed clear INPP5A loss,whereas control tissues showed no detectable loss of FISH signal (FIG.3). Normal metaphase spread is provided as a reference, and two copiesof INPP5A are seen in normal keratinocytes (FIG. 3, left and right-toppanels) with INPP5A signal in red, but only one copy in SCC (FIG. 3,right-bottom panel).

The observed deletions of INPP5A in FIG. 3 represent a highly selected,nonrandom genetic event in SCC. Most of 191 deletions identified amongSCC samples occur only once, whereas samples of a smaller proportion areobserved as recurrent deletions, affecting more than one sample. Theregion of INPP5A is the single most frequently deleted segment in theinterrogated SCC genomes, as well as the only recurrent deletiondetected in five independent SCC samples. The core INPP5A deletion,characterized as the smallest area of overlap among the aberrationsharboring INPP5A deletions (FIG. 2), covers a genetic segment containing587,219 bp, of which 91,861 bp are in the INPP5A gene itself. Inaddition to INPP5A, this segment contains three other genes: GPR123,KNDC1, and VENTX. However, INPP5A is the only gene in this clusterrepeatedly affected by the copy number transition (the edge of theaberration), being affected in three of five samples harboring deletionof this region. Taken together, therefore, INPP5A gene deletions arehighly selected genetic events, rather than nonspecific bystander eventsin the context of the overall genomic instability of the SCC genome.

Example 2 INPP5A Protein Level is Frequently Reduced in Primary SCCTissues

Genomic aberrations detected by aCGH, such as gene deletions, canindicate a “tip of the iceberg” phenomenon, where loss of a gene on theDNA level is seen in a subset of tumors, whereas in remaining cases theimplicated gene may be deregulated by other mechanisms, including thoseaffecting its mRNA and protein products, and thus the loss of a gene onthe DNA level is not the only way by which loss of expressed protein canoccur.

To evaluate whether the genetic loss of INPP5A observed with aCGH mightbe similarly indicative of a more general phenomenon of INPP5A loss inSCC, INPP5A protein levels were examined in an independent cohort ofFFPE skin tissues by immunohistochemistry using a monoclonal antibody toINPP5A. A total of 71 archived SCC tumors were evaluated and comparedwith the matched normal skin from the same patient using thehistologically normal epidermis, immediately adjacent to the SCC tumoras control. Stained slides were evaluated using a standard scoringsystem based on the intensity of staining (0-3), with score of 0representing no staining and score of 3 as intense staining. If arelative difference in signal was observed between tissues beingcompared, it was recorded as a change in INPP5A protein level.

Detection of INPP5A by immunohistochemistry showed mainly diffusecytoplasmic signal. A comparison of INPP5A staining intensity betweenSCC tissues and matched normal epidermis identified three generalstaining patterns. The most prevalent pattern of expression, observed in51 of 71 (72%) examined tissues, manifested as a relative reduction ofINPP5A in SCC tissues when compared with matched normal skin (FIG. 4A).Only 20 of 71 (28%) examined tissues showed no difference in INPP5Astaining between the SCC and matched normal skin. Importantly, no singlecase was observed where INPP5A staining was more intense in SCC tumorthan in matched normal skin, further highlighting the specificity of theobserved pattern (Table 2. A section; FIG. 4B). Notably, 10 SCC lesionsexamined in this cohort were classified by pathology as SCCIS (cutaneoussquamous cell carcinomas in situ, a lesion with transepidermalkeratinocyte atypia, indicating that the entire epidermis is filled withatypical keratinocytes). Six of 10 of these SCCIS lesions showed reducedINPP5A immunohistochemical signal, indicating no significant differenceto the frequency detected in primary SCC in general. The observedreduction of INPP5A signal in SCC tissues is tumor specific, as thehistory of sun exposure and the extent of sun damage are comparablebetween the SCC lesion and the examined, adjacent normal epithelium usedas a control. Since loss of INPP5A was shown to be present in asignificant percentage of all stages of SCC, from precursor tometastatic disease, observing loss at AK, SCCIS, or local SCC indicatesa risk that the lesion where the loss is observed in may continue toevolve through the stages to eventually become metastatic SCC. Takentogether, a significantly higher frequency of INPP5A loss at the proteinlevel compared with loss at the DNA level indicates that gene deletionsmay represent only one mechanism by which loss of INPP5A proteinproduction can occur. There is also a sizable proportion of SCC tumorslikely achieve the same effect through deregulation of INPP5A by othermechanisms to reduce the INPP5A protein level.

TABLE 2 Detection of INPP5A protein levels at successive stages of SCCprogression INPP5A Staining Intensity Frequency A. Primary SCC comparedto matched normal skin Normal skin > Primary SCC 51/71 (72%) Normal skin= Primary SCC 20/71 (28%) Normal skin < Primary SCC 0/71 (0%) B. AKcompared to matched normal skin Normal skin > AK 9/26 (35%) Normal skin= AK 17/26 (65%) Normal skin < AK 0/26 (0%) C. Primary SCC compared tomatched metastatic tissues Primary SCC > Met 6/17 (35%) Primary SCC =Met 11/17 (65%) Primary SCC < Met 0/17 (0%)

Example 3 INPP5A Loss on the Protein Level is an Early Event in SCCDevelopment

To more precisely evaluate the timing of the reduction of INPP5A levelin the development of cutaneous SCC, a series of 26 AKs (actinickeratoses, lesions in which atypical keratinocytes do no fill theepidermis) were examined, the earliest step in SCC development. Usingimmunohistochemistry, as described above, INPP5A protein levels betweenthe AK lesions and adjacent normal epidermis were compared. A relativereduction of INPP5A in AK lesions was seen in 9 of 26 (35%) examinedtissues, whereas 17 of 26 (65%) examined tissues showed no difference inINPP5A levels between the AK and normal epidermis (Table 2. B.).

To test whether the reduction of INPP5A protein in AKs is caused bygenetic loss at the DNA level, such as seen in a subset of SCC tumors,FISH analysis was carried out, and no INPP5A gene deletion was detectedin any of the examined cases. Although a small lesion size and limitednumber of lesional nuclei available for analysis in some of the studiedAKs call for cautious interpretation of these results, it is importantto note that no single lesion showed evidence of a clonal populationwith uniform loss of INPP5A FISH signal, even in cases where such clonalloss was suggested by immunohistochemical data. This absence ofperturbations on DNA level in AKs may explain the relative paucity ofgene copy number aberrations at early stages of disease detected by aCGH(FIG. 1) and indicates that deregulation of INPP5A expression in theseprecursor lesions occurs mainly at the mRNA or protein level.

The less frequent reduction of INPP5A protein levels in AK than in SCClesions (35% versus 72%) is also informative and reflects a selectionthat favors progression of AK lesions with low INPP5A to the next stageof disease. FIG. 5 showed two representative lesions, each containing anarea of SCCIS (evident by full epidermal thickness neoplasia; right halfof each image), arising in association with an AK (partial epidermalthickness neoplastic change, consistent with AK; left half of eachimage). A pattern of INPP5A protein reduction in a subset of AKs,occurring in the form of strikingly demarcated regions of low INPP5Asignal is an evidence for clonally expanding populations of affectedcells within epidermis (FIGS. 5A and 5B). As SCCIS often arise withinthe preexisting lesions of AKs, this focal loss of INPP5A in AKsrepresents an early step toward a full oncogenic transformation alongthe spectrum of evolving epidermal neoplasia.

Example 4 Loss of INPP5A in Association with Progression to MetastaticDisease

The above data showed that deregulation of INPP5A levels as an earlyevent in the development of keratinocyte neoplasia, provides a selectiveadvantage in progression from AK to SCC. To assess a potential role ofINPP5A loss in the process of tumor maintenance and progressions, thepotential association of reduction of INPP5A level with the subsequentbiological step in SCC progression and development of metastatic diseasewas tested. INPP5A protein levels were evaluated in a selected cohort of17 patients with cutaneous SCC tumors that have subsequentlymetastasized, and INPP5A protein levels were also evaluated where bothprimary tumor tissue and matched regional metastatic tissue wereavailable for examination. Immunohistochemical analysis of these pairedtissues detected further reduction of INPP5A levels in the transitionfrom primary to metastatic SCC in 6 of 17 (35%) examined tissue pairs(Table 2. C).

Although the remaining 11 of 17 (65%) studied pairs show no further lossof INPP5A levels in transition from primary to metastatic disease, thereis no single case was identified where INPP5A staining was stronger inthe metastatic tissue than in the primary SCC tumor. This observationfurther highlighted the specificity of the observed INPP5A loss in SCCprogression. These data demonstrated that reduction of INPP5A levels,although an early event in development of SCC, also plays a role inprogression of SCC from primary to metastatic disease in a significantsubset of aggressive primary SCC tumors.

It was further noted during the INPP5A staining in the cohort of 17patients with metastatic SCCs, there were normal epidermis presentimmediately adjacent to the primary SCC tissue in 13 of 17 examinedprimary SCCs. Twelve of 13 (92%) of these aggressive primary SCC tumors,which is a strikingly high frequency, showed loss of INPP5A stainingwhen compared with the adjacent, normal epidermis. In comparison, inrandomly selected primary SCC tumors, 51 of 71 (72%) SCC tumors showedreduction of INPP5A protein levels by immunohistochemistry (FIG. 4;Table 2A). Thus, higher frequency of INPP5A loss in primary SCC tumorsthat have shown an aggressive clinical course (i.e., subsequentdevelopment of metastases) indicates more aggressive primary disease.INPP5A level is, therefore, of prognostic value in assessing the risk ofprogression in primary SCC tumors.

Example 5 IP6 Reinitiates Terminal Differentiation of Cells with ReducedINPP5A Expression

In normal skin, a gradient of increasing levels of INPP5A expression isseen as keratinocytes move from the basal proliferative layer and passthrough the various non-proliferative differentiation stages that end atthe cornified layer. In both AKs (actinic keratosis) and tumors, loss ofINPP5A expression may represent an escape mechanism that inappropriatelyallows these cells to retain high proliferative capacity. Ectopicexpression of INPP5A in SCC cell lines leads to apoptosis. INPP5A isknown to play a role in the synthesis of inositol hexaphosphate (IP6),and this compound exhibits effects on growth. Loss of INPP5A activityallows squamous cells to avoid cessation of growth and terminaldifferentiation. Exposure of cells that have lost INPP5A expression toIP6 reinitiates the terminal differentiation process. Specifically,exposure to IP6 induces apoptosis in SCC lines and primary,undifferentiated, proliferating keratinocytes in vitro. Therefore, thelevel of INPP5A expression identifies patients that would benefit fromIP6 therapy.

SCC4 cells were infected with lentiviral vectors that delivered eitherCMV-GFP (control) or CMV-INPP5A. The cells were evaluated two weekslater. A 5-fold induction of INPP5A expression over baseline wasconfirmed by Quantitative Real-Time PCR. The q Real-Time PCR was done onan ABI PRISM® 7000 device using an ABI kit per the manufacturer'sinstructions (Applied Biosystems Carlsbad, Calif.). The primer pair usedfor detecting INPP5A m-RNA expression level were5′-TTGCAGACTGTCCTTTGAC-3′ (SEQ ID NO: 3) and 5′-AAACCCTTCTCGAATCGCTGA-3′(SEQ ID NO: 4). FIG. 6 depicts increased apoptosis in a squamous cellcarcinoma cell line that carries a gene expression cassette thatexpresses INPP5A (B and D) compared to the same cell line carrying agene expression cassette that expresses an unrelated protein, greenfluorescent protein (A and C). The top panels of FIG. 6 depict CMV-GFPinfected cells and CMV-INPP5A transfected cells in culture 2 weeks afterinfection (20×). The bottom panels show CMV-GFP and CMV-INPP5A infectedSCC-4 cells after performance of TUNEL staining for cells undergoingapoptosis. Nuclei were stained blue using DAPI probe andapoptosis-positive cells are stained in red (lighter, more punctuate).Overexpression of INPP5A results in significant cell death by apoptosis.

IP6 is an inositol metabolite that is dependant upon INPP5A activity forits synthesis (See FIG. 7). Addition of exogenous IP6 inhibits the cellproliferation brought about by INPP5A loss and drives cells towards adifferentiated phenotype. In FIG. 8, the left panel (A) shows untreatedSCC-4 cells and the right panel (B) shows SCC-4 cells treated with IP6.Increased TUNEL staining (red) is seen in the IP6 treated cells.Therefore, treatment of SCC-4 cells with IP6 results in significant celldeath by apoptosis, similar to the result seen in FIG. 8 with theoverexpression of INPP5A.

IP6 is selective for cells with reduced INPP5A expression. Normal humankeratinocytes were harvested at various stages of confluency. Cellcultures were assessed by morphological evaluation of the cell andsegregated into distinct culture confluence intervals at or near theplateau phase of growth, generally; lower confluence (L), 70-80%; mediumconfluence (M), 80-90%, and higher confluence (H), >95%, INPP5A mRNAexpression at selected culture confluence levels was evaluated by qPCR.INPP5A mRNA expression by q-RTPCR as a function of % of confluence ofnormal human keratinocytes are provided in Table 3 and FIG. 9.

TABLE 3 INPP5A mRNA expression at selected confluence level ConfluenceINPP5A mRNA Fold Change 70% 1 90% 2.8 100%  3.4 4-d post 4.1

INPP5A protein level in cells at different confluence levels wasmeasured using Western Blot (FIG. 10 and FIG. 11). Cytokeratin-10 (CK10)and integrin β4 are two known markers associated with keratinocytedifferentiation. Keratinocyte differentiation level as a function ofincreased confluence was demonstrated by increased expression ofCytokeratin-10 (CK10) mRNA and decreased expression of integrin β4 mRNAas determined by qPCR using ABI TaqMan kits for detecting human KRT10and human ITGB4 mRNA and provided in Table 4. These results areconsistent with the expression patterns in keratinocyte differentiation.

TABLE 4 Expression of known markers of keratinocyte differentiation atselected confluence level Fold Induction mRNA NHK Confluence KRT10 ITGB470% 1.00 1.00 90% 6.34 1.51 100%  124.34 0.38 4-day post 5294.22 0.66

FIG. 12 and FIG. 13 depict reduced apoptosis in more highly confluentand differentiated keratinocytes. FIG. 12, left panel (A) shows 100%confluent keratinocytes that were untreated. and the right panel (B)shows 100% confluent keratinocytes treated with 2 mM IP6 for 24 hours.FIG. 13 left panel (A) shows 70% confluent keratinocytes that wereuntreated; and the right panel (D) shows 70% confluent keratinocytesthat were treated with 2 mM IP-6 for 24 hours. Greater apoptosisrelative to background was seen in the 70% confluent keratinocytes incomparison to the 100% confluent keratinocytes.

Example 6 INPP5A Cellular Characterization

INPP5A was delivered to cells in a controlled expression vector pTUNE, aderivative developed from a commercial vector (FIG. 14). Introduction ofINPP5A with controlled expression allows production of a small amount ofthe protein. However, in the head and neck cancer cell line Cal27, thesmall amount of the INPP5A protein expression was sufficient to reducemigration in a scratch assay by 40%. A comparison of a 24-hour assay inthe absence or presence of INPP5A in the Cal27 line is shown by thescratch assay shown in FIG. 15. While CAL27 did not show a growthreduction after INPP5A transfection, it did show a marked loss inmotility (FIG. 15). A scratch assay shows fill-in of an approximately 1mm scratch in 8 hours for the unmodified CAL27 line, but no fill in over24 hours in the Cal-27+INPP5A transfectants. The growth rates of headand neck squamous cell carcinoma cell lines with and without the INPP5Aexpression plasmid were tested. Cell lines (Table 5) were plated in 12well plates. Cal-27 and SCC-15 were grown in DMEM media, and SCC-9, -4,and -25 were grown in DMEM F12 media. All cell lines were grown to 70%confluence. 1.6 micrograms of AhdI linearized pTUNE INPP5A (Origene,Rockville, Md.) were transfected into the cell lines using lipofectamineand Optimem media (Invitrogen, Carlsbad, Calif.). After transfection,cells carrying the INPP5A bearing vector were selected with G418 at 1microgram/ml for CAL27 and at 0.5 microgram/ml for the remaining celllines. Cells were examined microscopically for outgrowth oftransformants and those cell lines not growing were examined with thevital fluorescent stain Vybrant Violet (Invitrogen, Carlsbad, Calif.) toobserve apoptotic nuclear decay. The results are provided in Table 5below.

TABLE 5 Effects of INPP5A expression on growth of selected cell lines:Cell Doubling time Doubling Line Control Time + INPP5A Comments Cal-271-2 days 1-2 days no change SCC-9 4-5 Days 4-5 days no change SCC-15 2-3days NA Slowly dying over 16 days post transfection (FIG. 18) SCC-25 5-6days NA Slowly dying over 12 days post transfection SCC-4 4-5 days NACells all died within 4 days of transfection

Note that in SCC15+INPP5A and SCC-25+INPP5A, a vital nuclear dye showspycnotic nuclei and apoptosis. FIG. 16 depicts SCC 15+INPP5A stainedwith Vybrant Violet, and the small, dark pycnotic nuclei were shown inapopotosis. Similar results were seen in SCC-25. Table 6 showed theq-Real-Time PCR results of INPP5A mRNA level in selected cell linesnormalized to ACTB, a control gene. The primer used for INPP5Aq-Real-Time PCR were 5′-TTGCAGACTGTCCTTTGAC-3′ (SEQ ID NO: 3) and5′-AAACCCTTCTCGAATCGCTGA-3′ (SEQ ID NO: 4). The primers used for ACTBq-Real-Time PCR was 5′-TCATGAAGTGTGACGTGGACATC-3′ (SEQ ID NO: 5) and5′-CAGGAGGAGCAATGATCTTGATCT-3′ (SEQ ID NO: 6).

TABLE 6 q-Real-Time PCR of INPP5A mRNA level in selected cell linesnormalized to ACTB Cell Line Critical Cycle INPP5A-ACTB % of ACTB CAL273.46 9.1% CAL27 + INPP5A 2.99 12.6% SCC25 3.44 9.2% SCC15 3.32 10.0%SCC9 3.80 7.2% SCC4 3.66 7.9%

Example 7 Dose-Dependent IP6 Treatment of Squamous Cell Carcinoma

The high frequency of loss of INPP5A in skin squamous cell carcinoma(SCC) and as well as its loss in sun damaged skin, an early precursor ofSCC, was demonstrated in above examples. As shown in FIG. 5 and FIG. 17,immunohistochemical staining for the presence of INPP5A demonstratedthat INPP5A normally appears at a higher and higher level askeratinocytes go through the various stages of differentiation in thetransition from actively growing precursor cells to the dead, cornifiedcells of the outer dermis. Without being bound by theory, one way thatINPP5A could be involved in this differentiation process is that it actsas a requisite enzyme in the formation of a small molecule involved incell signaling. As INPP5A mediates the dephosphorylation of the 5phospho group on either Ins(1,4,5)P3 or Ins(1,3,4,5)P4, the test in thisexample was carried out to determine whether restoring thephospho-inositol metabolites that are downstream of the INPP5A-mediatedstep to SCC cancer cell lines could lead to differentiation and death.

The diagram in FIG. 7 shows two inositol phosphate metabolic branchesresulting directly from the action of INPP5A. In this invention, IP6 wastested for activity against a number of squamous cell cancer linesderived from head and neck tumors (SCC-4 SCC-15 SCC-9), and both acolorectal adenocarcinoma (HT-29) and a transformed normal humanembryonic kidney cell line (HEK-293). Cultures of 600 cells for each ofthese lines in growth media were established in wells of a 384 wellplate, and then sets of four replicates per dose were treated by theaddition of no drug, 0.31 mM, 0.63 mM, 1.25 mM, 2.5 mM and 5 mM IP6.These treated and untreated cultures were allowed to grow for 72 hoursin a tissue culture incubator and then were treated with and equalvolume of Cell Titer Glow reagent, which lyses the cells and produceslight through a reaction utilizing the ATP available from the cells.

The proportion of cells present after treatment relative to the numberof cells in the untreated cells was shown in FIG. 18. As demonstrated,the cells showed a dose-dependent reduction in number, which is muchgreater for the SCC cells than for the colorectal adenocarcinoma or thetransformed human embryonic kidney cells. This data presented thatINPP5A's loss leads to a failure of cells produced by lessdifferentiated basal cells to cease proliferating and differentiate tofrom a liner layer and that this loss of regulation can to some extentbe corrected by supplying an inositol phosphate metabolite whosesynthesis normally requires the activity of INPP5A. Therefore thedetection of loss of INPP5A activity in tumors that are dependent onthis loss for their survival will prove useful in directing therapy forthese tumors.

We claim:
 1. A method of treating a tumor, the method comprising thestep of: contacting the tumor with an effective therapeutic amount of apharmaceutical composition comprising inositol hexaphosphate (IP6) or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein tumor is associated with skin cancer.
 3. The method of claim 1,wherein the tumor is squamous cell carcinoma, squamous cell carcinoma insitu, or actinic keratosis.
 4. The method of claim 1, wherein the tumoris suspect to be squamous cell carcinoma, squamous cell carcinoma insitu, or actinic keratosis.
 5. The method of claim 4, wherein the tumorhas an INPP5 deletion.
 6. The method of claim 1, wherein thepharmaceutical composition comprises at least one excipient.
 7. Themethod of claim 1, wherein the pharmaceutical composition is formulatedfor topical administration.
 8. The method of claim 7, wherein contactingthe tumor comprises topically administering the pharmaceuticalcomposition to a subject with the tumor.
 9. A method of treating skincancer, the method comprising the step of: administering to a subjectsuspect of having skin cancer an effective therapeutic amount of apharmaceutical composition comprising IP6 or a pharmaceuticallyacceptable salt thereof.
 10. The method of claim 9, wherein the skincancer is selected from the group consisting of squamous cell carcinomaor squamous cell carcinoma in situ.
 11. The method of claim 10, whereincells of the skin cancer have an INPP5 deletion.
 12. The method of claim9, wherein the pharmaceutical composition is formulated for topicaladministration.
 13. The method of claim 12, wherein the pharmaceuticalcomposition is formulated as at least one of a liquid solution, cream,paste, lotion, shake lotion, powder, emulsion, ointment, gel base, and atransdermal patch.
 14. The method of claim 9, wherein the pharmaceuticalcomposition comprises at least one excipient.
 15. The method of claim14, wherein the at least one excipient is selected from the listconsisting of petrolatum, lanolin, polyethylene glycols, beeswax,mineral oil, diluents such as water and alcohol, and emulsifiers andstabilizers, and one or more thickening agents.
 16. A method of treatinga subject with skin cancer or a condition that may progress to skincancer, the method comprising the step of: administering to the subjectan effective therapeutic amount of a pharmaceutical compositioncomprising IP6 or a pharmaceutically acceptable salt thereof.
 17. Themethod of claim 16, wherein the pharmaceutical composition comprises atleast one excipient selected from the list consisting of petrolatum,lanolin, polyethylene glycols, beeswax, mineral oil, diluents such aswater and alcohol, and emulsifiers and stabilizers, and one or morethickening agents.
 18. The method of claim 16, wherein thepharmaceutical composition is formulated for topical administration. 19.The method of claim 18, wherein the pharmaceutical composition isformulated as at least one of a liquid solution, cream, paste, lotion,shake lotion, powder, emulsion, ointment, gel base, and a transdermalpatch.
 20. The method of claim 16, wherein the skin cancer or conditionthat may progress to skin cancer is selected from the group consistingof squamous cell carcinoma, squamous cell carcinoma in situ, or actinickeratosis, and further where in the skin cancer or condition that mayprogress to skin cancer is associated with cells that have an INPP5deletion.